Genetic reprogramming of bacterial biofilms

ABSTRACT

Described herein are methods and compositions relating to engineered curli fibers, e.g. CsgA polypeptide. In some embodiments, the methods and compositions described herein relate to functionalized biofilms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 15/808,237, filed Nov. 9, 2017 which, in turn, is a continuationapplication of U.S. application Ser. No. 14/786,304, filed Oct. 22,2015, which issued as U.S. Pat. No. 9,815,871 on Nov. 14, 2017, which,in turn, is a national stage entry of PCT/US2014/035095, filed on Apr.23, 2014, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/814,908, filed on Apr. 23, 2013. Theentire contents of each of the foregoing applications are incorporatedherein by reference in their entirety.

TECHNICAL FIELD

The technology described herein relates to engineered polypeptides,bacteria comprising such polypeptides, and biofilms comprising saidbacterial cells.

BACKGROUND

In nature, most bacteria exist as biofilm communities, residing in aself-generated protective nanoscale scaffold of proteins, sugars,lipids, and extracellular DNA that defends against environmental rigors.Biofilm formation is essential for bacterial adhesion and colonizationof both natural and man-made surfaces. These highly evolvedextracellular matrices hold untapped potential as a beneficialnanobiotechnology engineering platform. There is a significant body ofwork that investigates the use of biofilms for beneficial purposes suchas wastewater treatment and biotransformations, but these efforts focuson the use of naturally occurring organisms that happen to have evolvedvarious desired qualities. Efforts to rationally engineer the structureof biofilms at the molecular level have been absent. To date, thereexists no robust and broad technology for the facile engineering ofbiofilm components.

SUMMARY

Described herein is a technology which permits the programming of an E.coli biofilm's functional properties by genetically appending functionalpeptide domains to the CsgA protein. After the new CsgA-peptide issecreted and assembled, the amyloid nanofiber network displays thepeptide in very high density on its surface. The biofilm's function isthen augmented according to the sequence of the displayed peptides. Itis demonstrated herein that functional peptide domains of variouslengths and secondary structures can be appended to CsgA withoutprecluding the formation of curli fibers. Lastly, it is demonstratedthat the peptide domains maintain their function in the context of thebiofilm after secretion and assembly.

In one aspect, described herein is an engineered CsgA polypeptide,comprising a CsgA polypeptide with a C-terminal display tag flanking theCsgA polypeptide; wherein the display tag comprises an activitypolypeptide and a linker sequence; wherein the linker sequence islocated N-terminal to the display polypeptide; and wherein the linkersequence comprises at least 6 amino acids. In some embodiments, thelinker sequence consists of glycine and serine residues. In someembodiments, the display tag and/or the activity polypeptide comprises apolypeptide selected from the group consisting of metal binding domain(MBD); SpyTag; graphene binding (GBP); carbon nanotube binding (CBP);gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and AFP8.

In one aspect, described herein is a nucleic acid sequence encoding theengineered CsgA polypeptide. In one aspect, described herein is a vectorcomprising the nucleic acid sequence encoding the engineered CsgApolypeptide. In one aspect, described herein is an engineered microbialcell comprising the vector, nucleic acid sequence, or engineered CsgApolypeptide. In one aspect, described herein is a biofilm comprising acell described herein. In one aspect, described herein is a biofilmproduced by culturing the cells described herein under conditionssuitable for the production of a biofilm.

In one aspect, described herein is a composition comprising anengineered CsgA polypeptide. In some embodiments, the compositioncomprises filaments comprising the engineered CsgA polypeptide. In someembodiments, the composition comprises a proteinaceous network. In someembodiments, the composition further comprises additional proteinaceousbiofilm components. In some embodiments, the composition furthercomprises a cell as described herein.

In one aspect, described herein is the use of a cell, composition, orbiofilm as described herein, to display a polypeptide within thebiofilm, with the composition, or on the cell surface. In one aspect,described herein is the use of a cell, composition, or biofilm asdescribed herein, in an application selected from the group consistingof biocatalysis; industrial biocatalysis; immobilized biocatalysis;chemical production; filtration; isolation of molecules from an aqueoussolution; water filtration; bioremediation; nanoparticle synthesis;nanowire synthesis; display of optically active materials; biosensors;surface coating; therapeutic biomaterial; biological scaffold;structural reinforcement of an object; and as a delivery system fortherapeutic agents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F depict a demonstration of the engineered biofilms describedherein. FIG. 1A depicts a diagram of the molecular engineering ofbacterial biofilms. FIG. 1B depicts, on the left, a schematic of theinsertion library of CsgA-MBD fusions (Nor C-terminus, with variousflexible linkers). On the right is an image of Congo Red plate assay ofinsertion library. FIG. 1C depicts a table of differentpeptide/polypeptide insertions into the C3 site. FIG. 1D depicts streaksof the various CsgA fusions onto Congo Red plates. Red coloration isindicative of curli formation. FIG. 1E depicts TEM of: wildtype curlinanofibers (+CsgA), top; Curli-MBD fusion, middle; and Curli-SpyTagfusion, bottom. Arrows indicate curli fibers. FIG. 1F depicts FE-SEMimages of wildtype curli nanofibers (+CsgA), top; and Curli-SpyTagfusion, bottom. Arrows indicate curli fibers.

FIGS. 2A-2C depict the functionalization of curli fibers. FIG. 2Adepicts a schematic of the attachment of proteins to thepeptide-displaying curli biofilms, including two components: 1) secretedCsgA monomers that self-assemble to form the curli nanofibers and 2) apeptide that can interact specifically and strongly with an interactingprotein domain (shown as a gray circle) to form a complex. By fusing theCsgA to the peptide at the C3 insertion site and coexpressing a fusionprotein consisting of a variable protein (purple hexagon) fused to theinteracting domain, the target protein can be displayed on engineeredcurli nanofibers. FIG. 2B depicts fluorescence microscopy images ofcurli biofilms after the addition of purified Venus-SC protein. Row 1:wild-type curli biofilms do not bind to the Venus-SC protein fusion. Row2: Curli biofilms displaying the ST peptide tag when exposed to aVenus-SC(E77Q) mutant that cannot for the covalent isopepetide bond. Row3: Only curli biofilms engineered to display the ST peptide tag showsspecific interaction with the Venus-SC. All images are fluorescencemicroscopy images, with the red channel showing bacterial DNA (SYTO-61)and the GFP channel indicating the presence of the Venus-SC fluorescentprotein reporter. FIG. 2C depicts the results of experiments with celllysate containing fusion proteins. Overlay images of SYTO-61 and GFPchannels indicates no binding of unpurified Venus-SC to wild-type curli(i), no binding of unpurified Venus-SC(E77Q) mutant to curli-STbiofilms, and to curli-ST (ii), and successful binding of unpurifiedVenus-SC mutant to curli-ST biofilms (iii).

FIG. 3 depicts a schematic of a screen of acceptable sites and linkersfor CsgA-peptide fusions. Genes encoding for the panel of CsgA chimericproteins with either N- or C-terminal fusion sitess. The MBD (SEQ ID NO:10) was fused to csgA either directly (N1, C1), with a short linker (N2,C2), or with a long linker (N3, C3) (SEQ ID NO: 13). Sec and N22sequences were maintained at the N-terminus to promote transport to theperiplasmic and extracellular spaces, respectively.

FIG. 4 depicts the modularity of C3 design for the design of BINDbiofilms displaying various functional peptides. A panel of peptidedomains was chosen to span a range of sizes, secondary structures, andfunctions. Each peptide was genetically fused to the C-terminus of CsgAthrough a 6-amino acid flexible linker. Spotted cultures were grown for48 hours on YESCA-CR plates.

FIGS. 5A-5D demonstrate the programmed adhesion of MBD-BIND biofilms to304L stainless steel. FIG. 5A depicts a photographic image depictingcells expressing no curli (left), wildtype CsgA (middle), and MBDdisplayed curli (right) spotted onto a 304L steel coupon, allowed todry, and then washed vigorously in water. Each of the spots on thesurface was imaged using SEM. Cells expressing no curli proteins (5B) orwild-type CsgA (5C) did not adhere to the steel surface, whereas thoseexpressing the CsgA-MBD fusion (5D) remained.

FIGS. 6A-6F demonstrate the covalent immobilization of full-lengthproteins onto curli biofilm using the SpyTag-SpyCatcher system. TEM andSEM images of PHL628 (AcsgA) strains expressing no curli (6A), wild-typeCsgA (FIGS. 6B-6C), and the SpyTag-BIND biofilms (FIGS. 6D-6E). FIG. 6Fdepicts biofilms grown on PLL-modified glass substrates and thenvisualized with a nucleic-acid stain (SYTO61) to determine the presenceof cells followed by treatment with SpyCatcher-Venus (GFP). Fluorescencemicroscopy of the biofilms reveals that only the proper combination ofCsgA-SpyTag and SpyCatcher-Venus results in significant proteinimmobilization. Biofilms expressing wt-CsgA or those treated with theSpyCatcher(EQ) mutant that is unable to form covalent bonds with SpyTagare not capable of immobilizing the fluorescent protein. All images arescaled identically (scale bar=5 μm) and are representative of the entirebiofilm-coated substrate surfaces.

FIGS. 7A-7B depict TEM images of cells expressing C3 mutation (FIG. 7A)and wt-CsgA (FIG. 7B) for comparison.

FIGS. 8A-8D depict a diagram of the creation of catalytic biofilms usingBIND. FIG. 8A depicts E. coli expressing CsgA fused to the 14 amino acidSpyTag (CsgA-ST). FIG. 8B depicts CsgA-ST assembled into amyloid fiberson the surface of the bacterium. When the bacteria form biofilms, curlifibers expressing ST create a polymer matrix around the cells. FIG. 8Cdemonstrates that this polymer matrix is covalently modified with anenzyme fused to SpyCatcher. FIG. 8D demonstrates that substrates toproduct conversion occurs on the high surface-area catalytic surface.

FIG. 9 depicts a SDS-PAGE gel of conjugation reaction between AmylaseSCand sheared curli fibers in PBS. Single star denotes AmylaseSC+CsgA-STconjugate and double star denotes AmylaseSC. Lane 1) CsgA+AmylaseSCprecipitation fraction, 2) CsgA-ST+AmylaseSC precipitation fraction, 3)CsgA+AmylaseSC soluble fraction, 4) CsgA-ST+AmylaseSC soluble fraction.

FIGS. 10A-10B depict biofilms immobilized on 96-well filter plate. FIG.10A depicts CsgA WT expressing cells and FIG. 10B depicts CsgA-STexpressing cells visualized with fluorescence microscopy DAPI stain.Confocal microscopy shows bacteria in mostly mono and bilayers.Difference in the quantity of bacteria is due to normalizing the cellseeding to curli rather than biomass. Scale bar for confocal images is10 μm.

FIGS. 11A-11B demonstrate AmylaseSC immobilization onto biofilms. CsgAWT and CsgA-ST expressing biofilms are incubated with AmylaseSC for 1.5h. FIG. 11A depicts a graph of biofilms containing ˜4×10⁷ cellsincubated with 20-1500 pmol AmylaseSC. Dotted line shows one-sitesaturation binding fit. FIG. 11B depicts a graph of biofilms withincreasing cell count incubated with 750 pmol AmylaseSC. Activity isreported in mM product released for 100 uL reaction.

FIGS. 12A-12B demonstrate the activity of immobilized AmylaseSC withvarying pH. CsgA WT and CsgA-ST expressing biofilms are incubated withPBS pH 2-12 for 2 h. FIG. 12A depicts a graph of the activity ofbiofilms compared with AmylaseSC in solution. FIG. 12B depits a graph ofthe metabolic activity of cells shown relative to activity at pH 7.

FIGS. 13A-13D demonstrate the activity of biofilms in organic solvents.Biofilms functionalized with AmylaseSC are incubated in water-miscibleand immiscible organic solvents. FIG. 13A depicts a graph of theactivity of AmylaseSC on the biofilms post incubation in a panel ofsolvents. FIG. 13B depicts a graph of the activity in FIG. 13A plottedagainst the partition coefficient of the organic solvents. FIG. 13Cdepicts a graph of the activity of AmylaseSC on the biofilms in varyingsolvent fraction of miscible organic solvent. FIG. 13D depicts a graphof the metabolic activity of cells post incubation in the panel ofsolvents.

FIGS. 14A-14B demonstrate the stability of AmylaseSC versus wild-typeα-Amylase in solution. FIG. 14A depicts a graph of the activity of thetwo enzymes after a 30 minute exposure to a range of temperatures. FIG.14B depicts a graph of the activity after storage at 4° C., 25° C., 37°C. over 64 days.

FIGS. 15A-15B demonstrate in vitro enzyme kinetics. FIG. 15A depicts agraph of the activity of AmylaseSC with different concentrations ofcolorimetric substrate 4-nitrophenyl-a-D-maltopentaoside (pNPMP). FIG.15B depicts Michaelis-Menten analysis.

FIGS. 16A-16P demonstrate the genetic programming and modularity of theBIND system. FIG. 16A depicts a diagram demonstrating that in the BINDplatform, csgA cells heterologously express and secrete fusion proteinsconsisting of an amyloidogenic domain (CsgA) and a functional peptidedomain. This fusion protein self-assembles into an extracellular networkof amyloid nanofibers, resulting in a biofilm material with programmednon-natural functions. FIG. 16B depicts quantitative assessment of theCongo Red binding from quadruplicate YESCA-CR spotted cultures usingintensity quantitation (ImageJ™) measures the relative amyloid producedfor each CsgA-peptide fusion, normalized to wild-type CsgA. Arepresentative set of culture spots onto YESCA-CR agar is shown at thetop. FIGS. 16C-16P depict FE-SEM images of the peptide fusion BINDlibrary transformed into LSR10 (MC4100, csgA) cells with no CsgA (FIG.16C), wt-CsgA (FIG. 16D), and the BIND peptide panel (see Table 1): HIS(FIG. 16E), GBP (FIG. 16F), FLAG (FIG. 16G), CNBP (FIG. 16H), A3 (FIG.16I), CLP12 (FIG. 16J), QBP1 (FIG. 16K), SpyTag (FIG. 16L), MBD (FIG.16M), CT43 (FIG. 16N), AFP8 (FIG. 16O), and Mms6 (FIG. 16P). Scale bars,1 μm.

FIG. 17 depicts a three-dimensional protein model of the BIND systembased on the C3 insertion site. Self-assembling CsgA amyloid domains arederived from protein threading of the CsgA sequence onto an AgfAhomology model. An example peptide domain, SpyTag (see Table 1), isshown in and the 6-residue flexible linker. This peptide structure waspredicted using PepFold and all structural manipulation performed inPyMol.

FIGS. 18A-18E demonstrate that BIND biofilms can be programmed to adhereto substrates or biotemplate nanoparticles. Adhesion of PHL628 csgAcells expressing no curli, wild-type CsgA, and CsgAMBD was tested byspotting grown cultures onto a 304L steel coupon and incubating for 48hours. Cells expressing no curli proteins (FIG. 18A) or wild-type CsgA(FIG. 18B) did not adhere to the steel surface, unlike those expressingthe MBD-BIND system (FIG. 18C); scale bars, 10 μm. The inset in (FIG.18C) shows the biofilm-modified steel surface, scale bar, 1 μm. Silvernanoparticles were template by A3-BIND biofilms incubated in aqueousAgNO3. PHL628 csgA cells expressing either wildtype CsgA or CsgA-A3 wereanalyzed by TEM after incubation in 147 mM AgNO3 for 4 hours (FIGS. 18Dand 18E).

FIGS. 19A-19G demonstrate covalent immobilization of full-lengthproteins onto curli biofilm. FIG. 19A depicts a schematic showing theprotein BIND immobilization strategy which uses an isopeptide bondforming splitprotein S. pyogenes FbaB adhesin system (24) to covalentlyattach proteins fused to the SpyCatcher domain onto BIND biofilmsdisplaying the 13-residue SpyTag. FIGS. 19B-19G depict TEM and FE-SEMimages of PHL628 csgA strains expressing no curli (FIGS. 19B and 19E),wild-type CsgA (FIGS. 19C and 19F), and the SpyTag-BIND biofilms (FIGS.19D and 19G). Scale bars, 1 μm.

FIGS. 20A-20N depict TEM images of the peptide fusion BIND librarytransformed into LSR10 (MC4100, csgA) cells with no CsgA (FIG. 20A),wt-CsgA (FIG. 20B), and the BIND peptide panel (see Table 1): HIS (FIG.20C), GBP (FIG. 20D), FLAG (FIG. 20E), CNBP (FIG. 20F), A3 (FIG. 20G),CLP12 (FIG. 20H), QBP1 (FIG. 20I), SpyTag (FIG. 20J), MBD (FIG. 20K),CT43 (FIG. 20L), AFP8 (FIG. 20M), and Mms6 (FIG. 20N). Scale bars, 1 μm.

FIGS. 21A-21B demonstrate immunogold TEM of FLAG-tagged BIND andwildtype CsgA nanofibers. FIG. 21A depicts LSR10 cells expressingFLAG-BIND and FIG. 21B depicts wildtype curli probed using an anti-FLAGprimary antibody and a 15-nm gold nanoparticle-labeled secondaryantibody. Samples were blocked with 0.1% BSA in PBS and washed with0.01% BSA in PBS before staining with 1% uranyl formate and imaged byTEM. Scale bars, 500 nm.

FIG. 22 depicts images demonstrating that FLAG-BIND biofilms occur asextensive 2D amyloid sheets. SEM images of meshlike sheets formed byLSR10 cells transformed with CsgA-FLAG are shown at variousmagnifications, filtered onto Nuclepore membranes. The arrows in eachimage points to the leading edge of the amyloid film.

DETAILED DESCRIPTION

Embodiments of the technology described herein relate to the inventors'discovery of how to functionalize bacterial biofilms. More specifically,the inventors have discovered CsgA, the major component of E. colibiofilms, can be engineered to comprise polypeptides having a functionor activity which provide the biofilm comprising the engineered CsgAwith a new property or activity.

In one aspect, the technology described herein relates to an engineeredCsgA polypeptide, comprising a CsgA polypeptide with a C-terminaldisplay tag flanking the CsgA polypeptide. The display tag comprises anactivity polypeptide and a linker sequence, wherein the linker sequenceis located N-terminal to the display polypeptide and wherein the linkersequence comprises at least 6 amino acids. As used herein, “CsgA” (asdistinguished from an engineered CsgA polypeptide) refers to the majorstructural subunit of curli. The sequences of CsgA and its homologs areknown in a number of species, e.g. the sequence of E. coli CsgA is known(NCBI Gene ID NO: 949055; SEQ ID NO: 1 (polypeptide)). In someembodiments, “CsgA” refers to E. coli CsgA. In some embodiments, “CsgA”refers to a polypeptide having at least 80% homology to SEQ ID NO: 1(e.g. 80% or greater homology, 90% or greater homology, or 95% orgreater homology), e.g. naturally occurring mutations or variants ofCsgA, homologs of CsgA, or engineered mutations or variants of CsgA.

As used herein, an “engineered CsgA polypeptide” refers to a polypeptidecomprising a C-terminal display tag flanking the CsgA polypeptide, e.g.a polypeptide display tag located on the c-terminus of a CsgApolypeptide. In some embodiments, the display tag is located on theC-terminus of a CsgA polypeptide of SEQ ID NO: 1. The display tag flanksthe CsgA polypeptide, i.e., the entirety of the CsgA polypeptide islocated on the N-terminus of the display tag, e.g. the display tag doesnot interrupt the sequence of the CsgA polypeptide.

As used herein a “display tag” is a polypeptide engineered to be locatedat the C-terminus of a polypeptide comprising a CsgA polypeptide. Insome embodiments, a display tag comprises no more than 100 amino acids.In some embodiments, a display tag comprises no more than 50 aminoacids. In some embodiments, a display tag comprises no more than 40amino acids. In some embodiments, a display tag comprises no more than30 amino acids.

A display tag as described herein comprises, from N-terminus toC-terminus, a linker sequence and an activity polypeptide. A linkersequence is a polypeptide sequence of at least 6 amino acids. In someembodiments, the linker sequence comprises from about 6 amino acids toabout 50 amino acids. In some embodiments, the linker sequence comprisesfrom about 6 amino acids to about 100 amino acids. In some embodiments,the linker sequence comprises from about 30 amino acids to about 100amino acids. In some embodiments, the linker sequence comprises fromabout 40 amino acids to about 100 amino acids. In some embodiments, thelinker sequence comprises from about 50 amino acids to about 100 aminoacids. In some embodiments, the linker sequence comprises from about 6amino acids to about 30 amino acids. In some embodiments, the linkersequence comprises from about 20 amino acids to about 50 amino acids. Insome embodiments, the linker sequence comprises from about 30 aminoacids to about 50 amino acids. In some embodiments, the linker sequencecomprises from about 40 amino acids to about 50 amino acids. In someembodiments, the linker sequence comprises from about 6 amino acids toabout 20 amino acids. In some embodiments, the linker sequence comprisesfrom about 6 to about 10 amino acids. In some embodiments, the linkersequence comprises a flexible polypeptide, e.g a polypeptide not havinga rigid secondary and/or tertiary structure. In some embodiments, thelinker sequence comprises glycine and serine residues. In someembodiments at least 50% of the amino acids comprised by the linkersequence are glycine or serine residues, e.g. at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, or more areglycine or serine residues. In some embodiments, the linker sequenceconsists of glycine and serine residues.

As referred to herein, an “activity polypeptide” refers to a polypeptidehaving an activity or function, such that when it is present in abiofilm, it confers upon the biofilm a property, function, or activitywhich it did not have in the absence of the activity of the polypeptide.Accordingly, an activity polypeptide can be, e.g. an enzyme, apolypeptide that binds another molecule, a binding domain, a peptidethat is bound by another molecule (e.g. a ligand or epitope), or thelike. Examples of polypeptides for use as activity polypeptides include,but are not limited to Metal binding domain (MBD); SpyTag; graphenebinding (GBP); carbon nanotube binding (CBP); gold binding (A3); CT43;FLAG; Z8; E14; QBP1; CLP12; and AFP8. The sequences of these exemplaryembodiments are provided herein, e.g. in FIG. 1C and Table 1.

In some embodiments, the activity polypeptide, when present as part ofan engineered CsgA polypeptide, is functional. As used herein, apolypeptide is said to be “functional” or expressed as a “functional”polypeptide if the polypeptide retains at least about 50% of theactivity (e.g. enzymatic activity or binding activity) that it has as anisolated polypeptide. One of skill in the art can readily detectincreases in reaction products and/or detect decreases in reactionsubstrates, e.g. by mass spectroscopy (MS, including, e.g., MADLI/TOF,SELDI/TOF, LC-MS, GC-MS, HPLC-MS, etc., among others) or detectincreases or decrease in binding to a binding partner, e.g. byimmunoassays. In some embodiments, a functional activity polypeptide canretain at least 50% of the activity of the isolated polypeptide, e.g.50% or more of the activity, 60% or more of the activity, 75% or more ofthe activity, or 90% or more of the activity of the isolatedpolypeptide.

In some embodiments, the activity polypeptide can be a conjugationdomain. Such embodiments can permit immobilization of target proteins inthe biofilm, e.g., when the target protein is too large to be expressedas a fusion with CsgA. The conjugation domain present on the engineeredCsgA polypeptide can specifically bind to a partner conjugation domainpresent as part of the target protein, thereby incorporating the targetprotein into the biofilm. As used herein, “conjugation domain” refers toa polypeptide that can specifically bind to and/or be specifically boundby a partner conjugation domain, e.g. under conditions suitable forgrowth of a biofilm. A conjugation domain can be, e.g., about 100 aminoacids or less in size, about 75 amino acids or less in size, about 50amino acids or less in size, about 40 amino acids or less in size orsmaller. A partner conjugation domain can be about the same size as theconjugation domain or larger, e.g., a partner conjugation domain can beabout 4000 amino acids or less in size, about 3000 amino acids or lessin size, about 2000 amino acids or less in size, about 1000 amino acidsor less in size, about 500 amino acids or less in size, about 200 aminoacids or less in size, about 100 amino acids or less in size, about 75amino acids or less in size, about 50 amino acids or less in size, about40 amino acids or less in size, or smaller. In some embodiments, thebinding of the conjugation domain and partner conjugation domain iscovalent. Examples of conjugation domains are known in the art andinclude, but are not limited to, SpyTag; biotin acceptor peptide (BAP);biotin carboxyl carrier protein (BCCP); and a peptide comprising a LPXTGmotif. Similarly, partner conjugation domains are known in the art andinclude but are not limited to, respectively, SpyCatcher, streptavidin;streptavidin; and peptides comprising aminoglycine. Further discussionof conjugation systems comprising a conjugation domain and a partnerconjugation domain can be found, e.g., in Mao et al. J Am Chem Soc 2004126:2670-1; Zakeri et al. PNAS 2012 109:E690-E697; and Maeda et al. ApplEnviron Microbil 2008 74:5139-5145; each of which is incorporated byreference herein in its entirety. In some embodiments, an engineeredCsgA polypeptide comprising conjugation domain has the sequence of SEQID NO: 3 or is encoded by a polynucleotide having the sequence of SEQ IDNO: 2.

Where the activity polypeptide is a conjugation domain, the targetpolypeptide comprising the partner conjugation domain can furthercomprise a “functionalizing polypeptide.” As used herein, a“functionalizing polypeptide” refers to a polypeptide having an activityor function, such that when it is present in a biofilm, it confers uponthe biofilm a property, function, or activity which it did not have inthe absence of the polypeptide. A functionalizing polypeptide can be ofany size and is not part of the engineered CsgA polypeptide. Exemplaryfunctionalizing polypeptide can include, e.g. an enzyme, a polypeptidethat binds another molecule, an antibody or the like. In someembodiments, a polypeptide comprising a functionalizing polypeptide anda conjugation domain can further comprise an extracellular localizationtag, e.g. a sequence which will cause a cell expressing the polypeptideto secrete the polypeptide.

A functionalized engineered CsgA polypeptide or functionalized biofilmcan be provided by contacting an engineered CsgA polypeptide comprisinga conjugation domain (or a cell and/or biofilm comprising thatpolypeptide) with a polypeptide comprising the partner conjugationdomain. In some embodiments, the engineered CsgA polypeptide and thepolypeptide comprising the partner conjugation domain are maintained incontact for a period of time, i.e. the “binding step.” In someembodiments, the binding step is followed by a washing step, e.g. toremove excess unbound polypeptide.

In some embodiments, an engineered CsgA polypeptide comprising aconjugation domain is bound to (or binds) the partner conjugation domainin the presence of albumin (i.e. the “binding step”). In someembodiments, the albumin is BSA. In some embodiments, the albumin ispresent at about 0.1% to about 10%. In some embodiments, the albumin ispresent at about 0.5% to about 5%. In some embodiments, the albumin ispresent at about 1% to about 2%. In some embodiments, the binding stepis allowed to proceed for at least about 2 hours, e.g. about 2 hours ormore, about 6 hours or more, about 12 hours or more, or about 24 hoursor more. In some embodiments, the binding step is allowed to proceed inthe presence of albumin.

In some embodiments, the washing step proceeds for about 10 minutes toabout 6 hours. In some embodiments, the washing step proceeds for about30 minutes to about 3 hours. In some embodiments, the washing stepproceeds for about 90 minutes. In some embodiments, the polypeptides areagitated (e.g. shaken) during the washing step. In some embodiments, thewashing step comprises washing the polypeptides in a solution ofalbumin. In some embodiments, the albumin is BSA. In some embodiments,the albumin is present at about 0.01% to about 3%. In some embodiments,the albumin is present at about 0.1% to about 1%. In some embodiments,the albumin is present at about 0.3%. In some embodiments, the washingstep comprises 2 or more successive washes. In some embodiments, thewashing step comprises 3 successive washes.

As used herein, the term “specific binding” refers to a chemicalinteraction between two molecules, compounds, cells and/or particleswherein the first entity binds to the second, target entity with greaterspecificity and affinity than it binds to a third entity which is anon-target. In some embodiments, specific binding can refer to anaffinity of the first entity for the second target entity which is atleast 10 times, at least 50 times, at least 100 times, at least 500times, at least 1000 times or greater than the affinity for the thirdnontarget entity. A reagent specific for a given target is one thatexhibits specific binding for that target under the conditions of theassay being utilized.

In one aspect, described herein is a nucleic acid sequence encoding anengineered CsgA polypeptide as described herein. In one aspect,described herein is a vector comprising a nucleic acid sequence encodingan engineered CsgA polypeptide as described herein. The term “vector”,as used herein, refers to a nucleic acid construct designed for deliveryto a host cell or transfer between different host cells. As used herein,a vector can be viral or non-viral. Many vectors useful for transferringgenes into target cells are available, e.g. the vectors may be episomal,e.g., plasmids, virus derived vectors or may be integrated into thetarget cell genome, through homologous recombination or randomintegration. In some embodiments, a vector can be an expression vector.As used herein, the term “expression vector” refers to a vector that hasthe ability to incorporate and express heterologous nucleic acidfragments in a cell. An expression vector may comprise additionalelements, for example, the expression vector may have two replicationsystems, thus allowing it to be maintained in two organisms. The nucleicacid incorporated into the vector can be operatively linked to anexpression control sequence when the expression control sequencecontrols and regulates the transcription and translation of thatpolynucleotide sequence.

In some embodiments, a nucleic acid encoding an engineered CsgApolypeptide can be present within a portion of a plasmid. Plasmidvectors can include, but are not limited to, pBR322, pBR325, pACYC177,pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40,pBluescript II SK+/− or KS+/− (see “Stratagene Cloning Systems” Catalog(1993) from Stratagene, La Jolla, Calif., which is hereby incorporatedby reference), pQE, pIH821, pGEX, pET series (see Studier et. al., “Useof T7 RNA Polymerase to Direct Expression of Cloned Genes,” GeneExpression Technology, vol. 185 (1990), which is hereby incorporated byreference in its entirety).

As used herein, the term “viral vector” refers to a nucleic acid vectorconstruct that includes at least one element of viral origin and has thecapacity to be packaged into a viral vector particle. The viral vectorcan contain a transgenic gene in place of non-essential viral genes. Thevector and/or particle may be utilized for the purpose of transferringany nucleic acids into cells either in vitro or in vivo. Numerous viralvectors are known in the art and can be used as carriers of a nucleicacid into a cell, e.g. lambda vector system gill, gt WES.tB, Charon 4.

In some embodiments, the nucleic acid encoding an engineered CsgApolypeptide can be constitutively expressed. In some embodiments, thenucleic acid encoding an engineered CsgA polypeptide can be operablylinked to a constitutive promoter. In some embodiments, the nucleic acidencoding an engineered CsgA polypeptide can be inducibly expressed. Insome embodiments, the nucleic acid encoding an engineered CsgApolypeptide can be operably linked to an inducible promoter. In someembodiments, the nucleic acid encoding an engineered CsgA polypeptidecan be operably linked to a native CsgA promoter.

As described herein, an “inducible promoter” is one that ischaracterized by initiating or enhancing transcriptional activity whenin the presence of, influenced by, or contacted by an inducer orinducing agent than when not in the presence of, under the influence of,or in contact with the inducer or inducing agent. An “inducer” or“inducing agent” may be endogenous, or a normally exogenous compound orprotein that is administered in such a way as to be active in inducingtranscriptional activity from the inducible promoter. In someembodiments, the inducer or inducing agent, e.g., a chemical, a compoundor a protein, can itself be the result of transcription or expression ofa nucleic acid sequence (e.g., an inducer can be a transcriptionalrepressor protein), which itself may be under the control or aninducible promoter. Non-limiting examples of inducible promoters includebut are not limited to, the lac operon promoter, a nitrogen-sensitivepromoter, an IPTG-inducible promoter, a salt-inducible promoter, andtetracycline, steroid-responsive promoters, rapamycin responsivepromoters and the like. Inducible promoters for use in prokaryoticsystems are well known in the art, see, e.g. the beta.-lactamase andlactose promoter systems (Chang et al., Nature, 275: 615 (1978, which isincorporated herein by reference); Goeddel et al., Nature, 281: 544(1979), which is incorporated herein by reference), the arabinosepromoter system, including the araBAD promoter (Guzman et al., J.Bacteriol., 174: 7716-7728 (1992), which is incorporated herein byreference; Guzman et al., J. Bacteriol., 177: 4121-4130 (1995), which isincorporated herein by reference; Siegele and Hu, Proc. Natl. Acad. Sci.USA, 94: 8168-8172 (1997), which is incorporated herein by reference),the rhamnose promoter (Haldimann et al., J. Bacteriol., 180: 1277-1286(1998), which is incorporated herein by reference), the alkalinephosphatase promoter, a tryptophan (trp) promoter system (Goeddel,Nucleic Acids Res., 8: 4057 (1980), which is incorporated herein byreference), the PLtetO-1 and Plac/are-1 promoters (Lutz and Bujard,Nucleic Acids Res., 25: 1203-1210 (1997), which is incorporated hereinby reference), and hybrid promoters such as the tac promoter. deBoer etal., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983), which is incorporatedherein by reference.

An inducible promoter useful in the methods and systems as disclosedherein can be induced by one or more physiological conditions, such aschanges in pH, temperature, radiation, osmotic pressure, salinegradients, cell surface binding, and the concentration of one or moreextrinsic or intrinsic inducing agents. The extrinsic inducer orinducing agent may comprise amino acids and amino acid analogs,saccharides and polysaccharides, nucleic acids, protein transcriptionalactivators and repressors, cytokines, toxins, petroleum-based compounds,metal containing compounds, salts, ions, enzyme substrate analogs,hormones, and combinations thereof. In specific embodiments, theinducible promoter is activated or repressed in response to a change ofan environmental condition, such as the change in concentration of achemical, metal, temperature, radiation, nutrient or change in pH. Thus,an inducible promoter useful in the methods and systems as disclosedherein can be a phage inducible promoter, nutrient inducible promoter,temperature inducible promoter, radiation inducible promoter, metalinducible promoter, hormone inducible promoter, steroid induciblepromoter, and/or hybrids and combinations thereof. Appropriateenvironmental inducers can include, but are not limited to, exposure toheat (i.e., thermal pulses or constant heat exposure), various steroidalcompounds, divalent cations (including Cu2+ and Zn2+), galactose,tetracycline, IPTG (isopropyl-β-D thiogalactoside), as well as othernaturally occurring and synthetic inducing agents and gratuitousinducers.

Inducible promoters useful in the methods and systems as disclosedherein also include those that are repressed by “transcriptionalrepressors” that are subject to inactivation by the action ofenvironmental, external agents, or the product of another gene. Suchinducible promoters may also be termed “repressible promoters” where itis required to distinguish between other types of promoters in a givenmodule or component of the biological switch converters describedherein. Preferred repressors for use in the present invention aresensitive to inactivation by physiologically benign agent. Thus, where alac repressor protein is used to control the expression of a promotersequence that has been engineered to contain a lacO operator sequence,treatment of the host cell with IPTG will cause the dissociation of thelac repressor from the engineered promoter containing a lacO operatorsequence and allow transcription to occur. Similarly, where a tetrepressor is used to control the expression of a promoter sequence thathas been engineered to contain a tetO operator sequence, treatment ofthe host cell with tetracycline will cause the dissociation of the tetrepressor from the engineered promoter and allow transcription of thesequence downstream of the engineered promoter to occur.

In one aspect, described herein is an engineered microbial cellcomprising an engineered CsgA polypeptide and/or comprising a vector ornucleic acid encoding such a polypeptide.

In some embodiments, the engineered CsgA polypeptide can comprise anactivity polypeptide comprising a conjugation domain. In someembodiments, a cell encoding and/or comprising an engineered CsgApolypeptide can comprise an activity polypeptide comprising aconjugation domain can further encode and/or comprise a secondengineered polypeptide comprising a partner conjugation domain and afunctionalizing polypeptide. In some embodiments, described herein is apopulation of cells comprising two cell types, the first cell typeencoding and/or comprising an engineered CsgA polypeptide comprising anactivity polypeptide comprising a conjugation domain and the second celltype encoding and/or comprising a second engineered polypeptidecomprising a partner conjugation domain and a functionalizingpolypeptide. That is, it is contemplated herein that a single cell cancomprise a CsgA polypeptide with a conjugation domain and also comprisethe polypeptide which will bind to and/or be bound by that CsgApolypeptide or that a first cell can comprise a CsgA polypeptide with aconjugation domain and a second cell can comprise the polypeptide whichwill bind to and/or be bound by that CsgA polypeptide. It is furthercontemplated that an engineered CsgA polypeptide with a conjugationdomain can be contacted with a second polypeptide comprising a partnerconjugation domain and a functionalizing polypeptide, e.g. the secondpolypeptide can be produced (e.g. by a bacteria or eukaryotic cell)and/or synthesized (and optionally isolated or purified) and thenbrought in contact with the engineered CsgA polypeptide, e.g. when theCsgA polypeptide is present on a cell surface and/or present in abiofilm.

A bacterial cell of the methods and compositions described herein can beany of any species. Preferably, the bacterial cells are of a speciesand/or strain which is amenable to culture and genetic manipulation. Insome embodiments, the bacterial cell can be a gram-positive bacterialcell. In some embodiments, the bacterial cell can be a gram-negativebacterial cell. In some embodiments, the parental strain of thebacterial cell of the technology described herein can be a strainoptimized for protein expression. Non-limiting examples of bacterialspecies and strains suitable for use in the present technologies includeEscherichia coli, E. coli BL21, E. coli Tuner, E. coli Rosetta™, E. coliJM101, and derivatives of any of the foregoing. Bacterial strains forprotein expression are commercially available, e.g. EXPRESS™ CompetentE. coli (Cat. No. C2523; New England Biosciences; Ipswich, Mass.). Insome embodiments, the cell is an E. coli cell.

In some embodiments, the nucleic acid encoding an engineered CsgApolypeptide is comprised by a cell expressing wild-type CsgA. In someembodiments, the nucleic acid encoding an engineered CsgA polypeptide iscomprised by a cell with a mutation and/or deletion of the wild-typeCsgA gene, e.g. such that the cell does not express wild-type CsgA. Insome embodiments, the nucleic acid encoding an engineered CsgApolypeptide is introduced into a cell by homolgous recombination, e.g.such that the nucleic acid encoding an engineered CsgA polypeptidereplaces the wild-type CsgA gene in the cell.

In one aspect, described herein is a biofilm comprising an engineeredmicrobial cell comprising one or more engineered CsgA polypeptide and/orcomprising a vector or nucleic acid encoding such a polypeptides. Asused herein, a “biofilm” refers to a mass of microorganisms which canadhere or is adhering to a surface. A biofilm comprises a matrix ofextracellular polymeric substances, including, but not limited toextracellular DNA, proteins, glycopeptides, and polysaccharides. Thenature of a biofilm, such as its structure and composition, can dependon the particular species of bacteria present in the biofilm. Bacteriapresent in a biofilm are commonly genetically or phenotypicallydifferent than corresponding bacteria not in a biofilm, such as isolatedbacteria or bacteria in a colony.

In some embodiments, the technology described herein relates to abiofilm that is produced by culturing an engineered microbial cellcomprising an engineered CsgA polypeptide (and/or comprising a vector ornucleic acid encoding such a polypeptide) under conditions suitable forthe production of a biofilm. Conditions suitable for the production of abiofilm can include, but are not limited to, conditions under which themicrobial cell is capable of logarithmic growth and/or polypeptidesynthesis. Conditions may vary depending upon the species and strain ofmicrobial cell selected. Conditions for the culture of microbal cellsare well known in the art. Biofilm production can also be induced and/orenhanced by methods well known in the art, e.g. contacting cells withsubinhibitory concentrations of beta-lactam or aminoglycosideantibiotics, exposing cells to fluid flow, contacting cells withexogenous poly-N-acetylglucosamine (PNAG), or contacting cells withquorum sensing signal molecules. In some embodiments, conditionssuitable for the production of a biofilm can also include conditionswhich increase the expression and secretion of CsgA, e.g. by exogenouslyexpressing CsgD.

In some embodiments, the biofilm can comprise the cell which producedthe biofilm.

In some embodiments, described herein is a composition comprising anengineered CsgA polypeptide as described herein.

When expressed by a cell capable of forming curli, e.g. a cellexpressing CsgA, CsgB, CsgC, CsgD, CsgE, CsgF, and CsgG or some subsetthereof, CsgA units will be assembled to form curli filaments, e.g.polymeric chains of CsgA. In some embodiments, filaments of thepolypeptide can be present in the composition. In some embodiments, thefilaments can be part of a proteinaceous network, e.g. multiplefilaments which can be, e.g. interwoven, overlapping, and/or in contactwith each other. In some embodiments, the proteinaceous network cancomprise additional biofilm components, e.g. materials typically foundin an E. coli biofilm. Non-limiting examples of biofilm components caninclude biofilm proteins (e.g. FimA, FimH, Ag43, AidA, and/or TibA)and/or non-proteinaceous biofilm components (e.g. cellulose, PGA and/orcolonic acid). In some embodiments, the composition can further comprisean engineered microbial cell comprising an engineered CsgA polypeptideand/or comprising a vector or nucleic acid encoding such a polypeptide.

In one aspect, described herein is the use of a cell, composition, orbiofilm comprising an engineered CsgA polypeptide (and/or comprising avector or nucleic acid encoding such a polypeptide) to display apolypeptide, e.g. within the biofilm, within the composition, and/or onthe cell surface. As used herein, “display” refers to expressing thepolypeptide (e.g. as an activity polypeptide) in such a manner that itcan come in contact with the extracellular environment. A displayedpolypeptide can be capable of binding with a binding partner, catalyzingan enzymatic reaction, and/or performing any other activity which itwould perform as an isolated polypeptide.

It is contemplated herein that a polypeptide displayed within a biofilm(e.g. an activity polypeptide and/or functionalizing polypeptide) willretain more activity than a soluble version of that polypeptide. It iscontemplated herein that a polypeptide displayed within a biofilm (e.g.an activity polypeptide and/or functionalizing polypeptide) will retainmore activity than a soluble version of that polypeptide when exposed toactivity degrading conditions such as, e.g., high or low pH, organicsolvents, dessication, high or low temperature, radiation, etc.

In one aspect, described herein is the use of a cell, composition, orbiofilm comprising an engineered CsgA polypeptide (and/or comprising avector or nucleic acid encoding such a polypeptide), in an applicationselected from the group consisting of biocatalysis; industrialbiocatalysis; immobilized biocatalysis; chemical production; filtration;isolation of molecules from an aqueous solution; water filtration;bioremediation; nanoparticle synthesis; nanowire synthesis; display ofoptically active materials; biosensors; surface coating; therapeuticbiomaterial; biological scaffold; structural reinforcement of an object;and as a delivery system for therapeutic agents. Exemplary, non-limitingembodiments of such applications and specific activity polypeptides foruse therein are described in the Examples herein.

It is contemplated herein that a cell, composition and/or biofilm cancomprise multiple different engineered CsgA polypeptides, each of whichcomprises a different activity polypeptide, e.g. an engineered CsgApolypeptide comprising an enzymatic activity polypeptide and anengineered CsgA polypeptide comprising a binding domain activitypolypeptide. A cell, composition, and/or biofilm can comprise 1 or moreengineered CsgA polypeptides, e.g. 1, 2, 3, 4, 5, 6, or more engineeredCsgA polypeptides.

Exemplary, non-limiting embodiments of methods and compositionsdescribed herein follow:

BIND as a Biocatalytic Scaffold for the Display of Enzymes.

Biofilms are attractive as catalysts for a variety of biochemicaltransformations due to the their ability to withstand harsh conditions,their propensity for surface attachment and their scalability.

In general, biotransformation are catalyzed either by enzymes insideliving cells or by enzymes displayed on the cell surface. Bothapproaches have inherent limitations related to mass transfer andsolubility of substrates, in the case of whole cells, or related to thelimited surface area available on the cell membrane, in the case ofsurface display. Described herein is the development of technique calledBiofilm Integrated Nanofiber Display (BIND) that enables the rationaldesign of the biofilm extracellular matrix so that it can function as asubstrate for site-specific covalent surface immobilization of enzymes.α-Amylase fused to an attachment domain, SpyCatcher, was immobilizedonto E. coli biofilms displaying curli fibers with a capture domain,SpyTag. When compared to the free enzyme, the a-Amylase immobilized onthe biofilm surface was protected from harsh pH conditions and exposureto water immiscible organic solvents. This work lays the foundation fora new method of using the extracellular polymeric matrix of E. coli forcreating versatile and controllable biocatalytic surfaces.

BIND as a Living Coating for the Protection of Surfaces.

Surfaces used in many applications require protective coatings to renderthem resistant to wear, chemical degradation (i.e. corrosion), andfouling from chemical and biological sources. The BIND platform providesa way to create living surface coating materials that can be programmedto exhibit a range of protective functions for surfaces on which theengineered biofilms are immobilized: 1) strong adhesion to the surface,2) the ability to secrete soluble entities into the local environmentthat would prevent degradation or fouling, such as microbicides,reductants, etc., 3) the ability to secrete biopolymeric material tofill in cracks forming in the underlying substrate, or 4) the ability totemplate the growth of a mineral or other ordered material from aexogenously supplied building blocks.

Enzymatically-derived 13PDO can potentially be produced at a much lowercost than whole-cell fermentation processes, which require large amountsof media for every cycle and often produce metabolic side products,resulting in a complex mixture that must be processed to isolate thedesired product. The BIND platform technology will reduce the costbarriers typically associated with immobilized enzyme biocatalysis, byeliminating the multi-step purification and immobilization with a singleculturing step, and replacing costly synthetic scaffolds with anultra-stable self-produced scaffold. The metabolic production of 13PDOby microorganisms from glycerol proceeds from 2 enzymatic step. Thefirst step is the enzymatic dehydration of glycerol by Glyceroldehydratase (Gdh) to produce 3-hydroxypropionaldehyde (3HPA). The 3HPAis in turn reduced to 13PDO by an oxidoreductase, 1,3-propanedioldehydrogenase (Pdh). Both Gdh and Pdh have been studied extensively,with the best studied from Klebsiella pneumonia, a 1,3-PDO producingmicrobe. The DuPont process uses K. pneumonia genes cloned into E. colifor their industrial-scale fermentation process.

In some embodiments, a functionalizing polypeptide can comprise aglycerol dehydratase. In some embodiments, a functionalizing polypeptidecan comprise a K. pneumonia glycerol dehydratase. In some embodiments, afunctionalizing polypeptide can comprise a propanediol dehydrogenase. Insome embodiments, a functionalizing polypeptide can comprise a K.pneumonia propanediol dehydrogenase.

Biorecovery of Valuable Metals or Removal of Metal Pollutants UsingBIND.

Metal removal and recovery are highly relevant for a number ofindustries, including mining, recycling, and water treatment. The BINDplatform provides a method to display engineered binding proteins withhigh affinity and selectivity for specific metal ions and particles. TheBIND platform represents a significant advancement over otherbiosorption techniques because it is able to display full-lengthproteins in high density and in a scalable manner. The use ofrecombinant proteins for these applications would be cost-prohibitive inmost cases because of the production and purification protocolsnecessary to generate them. Contemplated herein are biofilm-basedmaterials that can serve as a separations medium for metal ions andparticles.

Engineering of Probiotic E. coli to Synthesize Therapeutic Biofilms.

Contemplated herein are biofilm-based materials that are suitable foruse inside the body. The BIND platform provides a means to program theadhesion of the biofilm-based material to biological tissues. Suchmaterials would be able to control the residence time and localizationof the biofilms inside the body. These materials would have thecapability of establishing themselves at a specified location, alteringthe properties of the biological tissue through direct interaction ofthe curli nanofibers with the tissue, and secreting soluble biomoleculesto alter local biological processes.

For convenience, the meaning of some terms and phrases used in thespecification, examples, and appended claims, are provided below. Unlessstated otherwise, or implicit from context, the following terms andphrases include the meanings provided below. The definitions areprovided to aid in describing particular embodiments, and are notintended to limit the claimed invention, because the scope of theinvention is limited only by the claims. Unless otherwise defined, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. If there is an apparent discrepancy between the usageof a term in the art and its definition provided herein, the definitionprovided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected here.

The terms “increased”, “increase”, “enhance”, or “activate” are all usedherein to mean an increase by a statically significant amount. In someembodiments, the terms “increased”, “increase”, “enhance”, or “activate”can mean an increase of at least 10% as compared to a reference level,for example an increase of at least about 20%, or at least about 30%, orat least about 40%, or at least about 50%, or at least about 60%, or atleast about 70%, or at least about 80%, or at least about 90% or up toand including a 100% increase or any increase between 10-100% ascompared to a reference level, or at least about a 2-fold, or at leastabout a 3-fold, or at least about a 4-fold, or at least about a 5-foldor at least about a 10-fold increase, or any increase between 2-fold and10-fold or greater as compared to a reference level.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably herein to designate a series of amino acid residues,connected to each other by peptide bonds between the alpha-amino andcarboxy groups of adjacent residues. The terms “protein”, and“polypeptide” refer to a polymer of amino acids, including modifiedamino acids (e.g., phosphorylated, glycated, glycosylated, etc.) andamino acid analogs, regardless of its size or function. “Protein” and“polypeptide” are often used in reference to relatively largepolypeptides, whereas the term “peptide” is often used in reference tosmall polypeptides, but usage of these terms in the art overlaps. Theterms “protein” and “polypeptide” are used interchangeably herein whenreferring to a gene product and fragments thereof. Thus, exemplarypolypeptides or proteins include gene products, naturally occurringproteins, homologs, orthologs, paralogs, fragments and otherequivalents, variants, fragments, and analogs of the foregoing.

A “variant,” as referred to herein, is a polypeptide substantiallyhomologous to a native or reference polypeptide, but which has an aminoacid sequence different from that of the native or reference polypeptidebecause of one or a plurality of deletions, insertions or substitutions.Polypeptide-encoding DNA sequences encompass sequences that comprise oneor more additions, deletions, or substitutions of nucleotides whencompared to a native or reference DNA sequence, but that encode avariant protein or fragment thereof that retains the relevant biologicalactivity relative to the reference protein. As to amino acid sequences,one of skill will recognize that individual substitutions, deletions oradditions to a nucleic acid, peptide, polypeptide, or protein sequencewhich alters a single amino acid or a small percentage, (i.e. 5% orfewer, e.g. 4% or fewer, or 3% or fewer, or 1% or fewer) of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid. It is contemplated that some changes canpotentially improve the relevant activity, such that a variant, whetherconservative or not, has more than 100% of the activity of a wildtype ornative polypeptide, e.g. 110%, 125%, 150%, 175%, 200%, 500%, 1000% ormore.

One method of identifying amino acid residues which can be substitutedis to align, for example, CsgA from E. coli to a CsgA polypeptide fromother species. Alignment can provide guidance regarding not onlyresidues likely to be necessary for function but also, conversely, thoseresidues likely to tolerate change. Where, for example, an alignmentshows two identical or similar amino acids at corresponding positions,it is more likely that that site is important functionally. Where,conversely, alignment shows residues in corresponding positions todiffer significantly in size, charge, hydrophobicity, etc., it is morelikely that that site can tolerate variation in a functionalpolypeptide. Such alignments are readily created by one of ordinaryskill in the art, e.g. created using the default settings of thealignment tool of the BLASTP program, freely available on the world wideweb at http://blast.ncbi.nlm.nih.gov/. Furthermore, homologs of anygiven polypeptide or nucleic acid sequence can be found using BLASTprograms, e.g. by searching freely available databases of sequence forhomologous sequences, or by querying those databases for annotationsindicating a homolog (e.g. search strings that comprise a gene name ordescribe the activity of a gene). Such databases can be found, e.g. onthe world wide web at http://blast.ncbi.nlm.nih.gov/.

The variant amino acid or DNA sequence can be at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or more,identical to a native or reference sequence. The degree of homology(percent identity) between a native and a mutant sequence can bedetermined, for example, by comparing the two sequences using freelyavailable computer programs commonly employed for this purpose on theworld wide web. The variant amino acid or DNA sequence can be at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or more,similar to the sequence from which it is derived (referred to herein asan “original” sequence). The degree of similarity (percent similarity)between an original and a mutant sequence can be determined, forexample, by using a similarity matrix. Similarity matrices are wellknown in the art and a number of tools for comparing two sequences usingsimilarity matrices are freely available online, e.g. BLASTp, withdefault parameters set.

In some embodiments, the variant is a conservative substitution variant.Variants can be obtained by mutations of native nucleotide sequences,for example. A “variant,” as referred to herein, is a polypeptidesubstantially homologous to a native or reference polypeptide, but whichhas an amino acid sequence different from that of the native orreference polypeptide because of one or a plurality of deletions,insertions or substitutions. Polypeptide-encoding DNA sequencesencompass sequences that comprise one or more additions, deletions, orsubstitutions of nucleotides when compared to a native or reference DNAsequence, but that encode a variant protein or fragment thereof thatretains the relevant biological activity relative to the referenceprotein. As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters a single aminoacid or a small percentage, (i.e. 5% or fewer, e.g. 4% or fewer, or 3%or fewer, or 1% or fewer) of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid. Itis contemplated that some changes can potentially improve the relevantactivity, such that a variant, whether conservative or note, has morethan 100% of the activity of the wildtype enzyme, e.g. 110%, 125%, 150%,175%, 200%, 500%, 1000% or more.

The degree of similarity (percent similarity) between an original and amutant sequence can be determined, for example, by using a similaritymatrix. Similarity matrices are well known in the art and a number oftools for comparing two sequences using similarity matrices are freelyavailable online, e.g. BLASTp, (available on the world wide web athttp://blast.ncbi.nlm.nih.gov) with default parameters set. A givenamino acid can be replaced by a residue having similar physiochemicalcharacteristics, e.g., substituting one aliphatic residue for another(such as Ile, Val, Leu, or Ala for one another), or substitution of onepolar residue for another (such as between Lys and Arg; Glu and Asp; orGln and Asn). Other such conservative substitutions, e.g., substitutionsof entire regions having similar hydrophobicity characteristics, arewell known. Polypeptides comprising conservative amino acidsubstitutions can be tested in any one of the assays described herein toconfirm that a desired apoptotic activity of a native or referencepolypeptide is retained. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. Suchconservatively modified variants are in addition to and do not excludepolymorphic variants, interspecies homologs, and alleles consistent withthe disclosure. Typically conservative substitutions for one anotherinclude: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)). Any cysteine residue not involved inmaintaining the proper conformation of the polypeptide also can besubstituted, generally with serine, to improve the oxidative stabilityof the molecule and prevent aberrant crosslinking. Conversely, cysteinebond(s) can be added to the polypeptide to improve its stability orfacilitate oligomerization.

As used herein, the term “nucleic acid” or “nucleic acid sequence”refers to any molecule, preferably a polymeric molecule, incorporatingunits of ribonucleic acid, deoxyribonucleic acid or an analog thereof.The nucleic acid can be either single-stranded or double-stranded. Asingle-stranded nucleic acid can be one nucleic acid strand of adenatured double-stranded DNA. Alternatively, it can be asingle-stranded nucleic acid not derived from any double-stranded DNA.In one aspect, the nucleic acid can be DNA. In another aspect, thenucleic acid can be RNA. Suitable nucleic acid molecules are DNA,including genomic DNA or cDNA. Other suitable nucleic acid molecules areRNA, including mRNA.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean±1%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the method or composition, yet open to the inclusion ofunspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of thisdisclosure, suitable methods and materials are described below. Theabbreviation, “e.g.” is derived from the Latin exempli gratia, and isused herein to indicate a non-limiting example. Thus, the abbreviation“e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can befound in The Encyclopedia of Molecular Biology, published by BlackwellScience Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X,published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321);Kendrew et al. (eds.), Molecular Biology and Biotechnology: aComprehensive Desk Reference, published by VCH Publishers, Inc., 1995(ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009,Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed usingstandard procedures, as described, for example in Sambrook et al.,Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (2001); Davis et al.,Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc.,New York, USA (1995); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds.,Academic Press Inc., San Diego, USA (1987); and Current Protocols inProtein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley andSons, Inc.), which are all incorporated by reference herein in theirentireties.

Other terms are defined herein within the description of the variousaspects of the invention.

All patents and other publications; including literature references,issued patents, published patent applications, and co-pending patentapplications; cited throughout this application are expresslyincorporated herein by reference for the purpose of describing anddisclosing, for example, the methodologies described in suchpublications that might be used in connection with the technologydescribed herein. These publications are provided solely for theirdisclosure prior to the filing date of the present application. Nothingin this regard should be construed as an admission that the inventorsare not entitled to antedate such disclosure by virtue of priorinvention or for any other reason. All statements as to the date orrepresentation as to the contents of these documents is based on theinformation available to the applicants and does not constitute anyadmission as to the correctness of the dates or contents of thesedocuments.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. For example, while methodsteps or functions are presented in a given order, alternativeembodiments may perform functions in a different order, or functions maybe performed substantially concurrently. The teachings of the disclosureprovided herein can be applied to other procedures or methods asappropriate. The various embodiments described herein can be combined toprovide further embodiments. Aspects of the disclosure can be modified,if necessary, to employ the compositions, functions and concepts of theabove references and application to provide yet further embodiments ofthe disclosure. Moreover, due to biological functional equivalencyconsiderations, some changes can be made in protein structure withoutaffecting the biological or chemical action in kind or amount. These andother changes can be made to the disclosure in light of the detaileddescription. All such modifications are intended to be included withinthe scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure.

The technology described herein is further illustrated by the followingexamples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be definedaccording to any of the following numbered paragraphs:

-   -   1. An engineered CsgA polypeptide, comprising a CsgA polypeptide        with a C-terminal display tag flanking the CsgA polypeptide;        -   wherein the display tag comprises an activity polypeptide            and a linker sequence;        -   wherein the linker sequence is located N-terminal to the            display polypeptide; and        -   wherein the linker sequence comprises at least 6 amino            acids.    -   2. The polypeptide of paragraph 1, wherein the linker sequence        consists of glycine and serine residues.    -   3. The polypeptide of any of paragraphs 1-2, wherein the display        tag and/or the activity polypeptide comprises a polypeptide        selected from the group consisting of:        -   Metal binding domain (MBD); SpyTag; graphene binding (GBP);            carbon nanotube binding (CBP); gold binding (A3); CT43;            FLAG; Z8; E14; QBP1; CLP12; and AFP8.    -   4. The polypeptide of any of paragraphs 1-2, wherein the        activity polypeptide comprises a conjugation domain.    -   5. The polypeptide of paragraph 4, wherein the conjugation        domain is selected from the group consisting of:        -   SpyTag; biotin acceptor peptide (BAP); biotin carboxyl            carrier protein (BCCP); and a peptide comprising a LPXTG            motif.    -   6. A nucleic acid sequence encoding the polypeptide of any of        paragraphs 1-5.    -   7. A vector comprising the nucleic acid sequence of paragraph 6.    -   8. An engineered microbial cell comprising the vector, nucleic        acid sequence, or polypeptide of any of paragraphs 1-7.    -   9. The cell of paragraph 7, wherein the cell expresses an        engineered CsgA polypeptide comprising an activity polypeptide        comprising a conjugation domain.    -   10. The cell of paragraph 9, wherein the cell further comprises        a nucleic acid sequence encoding a functionalizing polypeptide        comprising a partner conjugation domain.    -   11. A population of cells comprising a first cell type and a        second cell type, wherein the first cell type is a cell of        paragraph 9 and the second cell type comprises a nucleic acid        sequence encoding a functionalizing polypeptide comprising a        partner conjugation domain.    -   12. A biofilm comprising the cell of any of paragraphs 8-11.    -   13. A biofilm produced by culturing the cells of any of        paragraphs 8-11 under conditions suitable for the production of        a biofilm.    -   14. The biofilm of paragraph 13, comprising the cells of any of        paragraphs 8-11.    -   15. A composition comprising the polypeptide of any of        paragraphs 1-6.    -   16. The composition of paragraph 15, wherein the composition        comprises filaments comprising the polypeptide of any of        paragraphs 1-6.    -   17. The composition of any of paragraphs 15-17, comprising a        proteinaceous network.    -   18. The composition of any of paragraphs 15-18, wherein the        composition further comprises additional proteinaceous biofilm        components    -   19. The composition of any of paragraphs 15-19, further        comprising the cell of any of paragraphs 8-11.    -   20. The use of the cell, composition, or biofilm of any of        paragraphs 8-19, to display a polypeptide within the biofilm,        with the composition, or on the cell surface.    -   21. The use of the cell, composition, or biofilm of any of        paragraphs 8-19, in an application selected from the group        consisting of:        -   biocatalysis; industrial biocatalysis; immobilized            biocatalysis; chemical production; filtration; isolation of            molecules from an aqueous solution; water filtration;            bioremediation; nanoparticle synthesis; nanowire synthesis;            display of optically active materials; biosensors; surface            coating; therapeutic biomaterial; biological scaffold;            structural reinforcement of an object; and as a delivery            system for therapeutic agents.

EXAMPLES Example 1

Described herein is a method to genetically modify the majorproteinaceous component of bacterial biofilms to display functionalpeptides, thus reprogramming biofilms for a wide variety of beneficialapplications including but not limited to industrial biocatalysis,bioremediation, bioenergy, materials templating, and biosensing. Thistechnology is based on the curli system of E. coli, which consists ofcell-surface anchored amyloid fibrils that are made from theself-assembly of a secreted protein (FIGS. 1A-1F). The technologydescribed herein demonstrates that functional peptide domains can beappended to the secreted protein so that, once it is assembled, theamyloid fibril network exhibits augmented functionality (e.g., bindingto various biological and chemical entities). Furthermore, thisextracellular matrix-peptide display technology can be adapted for theimmobilization of any protein.

Results

Fusion Domains can be Displayed on Curli Nanofibers.

Many microbes produce biofilms as an extracellular matrix material tocolonize various surfaces and protect themselves from environmentalstresses. One of the major structural components of these biofilms isnanoscale fibers composed of proteins. The bacteria secrete theseproteins into the extracellular milieu, where the protein monomersspontaneously self-assemble into a polymeric chain which is anchored onthe cell surface. These protein nanofibers are known to exhibitamyloidogenic structural characteristics and are extremely robust. Theyhave been shown to impart a number of evolutionary advantages, includingmediating adhesion to surfaces, invasion of host cells, and sequesteringtoxic metals.¹⁻³ Most current biofilm research is focused on inhibitingor dispersing biofilms deleterious to human health. The formation ofbiofilms as an adhesion and persistence mechanism on almost any surfaceposes a great risk for infection in the biomedical and food industries⁴.Described herein is a platform technology which utilizes biofilms forbeneficial applications. It is demonstrated herein that functionalpeptides can be engineered into biofilms by the successful expressionand secretion of genetic fusions of fimbriae-forming nanofibers such ascurli.

Key aspects of the technology described herein can include, but are notlimited to: the ability to genetically program various physical,chemical, and biochemical functionalities into the protein structuralcomponent of a biofilm; a biofilm composed of curli nanofibers or anysuch similar extracellular self-assembling protein-based amyloid fibers;an engineered unit of the biofilm nanofiber composed of aself-assembling protein domain containing one or more curli unit orself-assembling domains, a spacer domain of variable length, and apeptide “activity” domain of arbitrary length on the C-terminus of theprotein; the N-terminus of the protein can optionally additionallycomprise various domains that allow periplasmic localization and/orprotein secretion into the extracellular space; the peptide activitydomain can be any peptide that allows for substrate adhesion, thebinding to any biomolecule or chemical, has self-contained catalyticactivity, is involved in catalytic activity in coordination with anexternally localized protein, can template inorganic structures, caninduce physiological responses in cells, can bind to ions in solution,can self-polymerize or polymerize with other molecules, is opticallyactive, confers electrical conductivity, and/or leads tostimulus-responsive behavior. In some embodiments, proteins can beimmobilized on the engineered curli biofilm by expressing on the curlinanofibers a peptide tag that specifically interacts with a proteindomain to form a covalent or non-covalent complex. Any target proteinfused to this interacting protein domain can thus be displayed on thebiofilms by exposing the engineered curli-peptide tag biofilms to thefusion protein. This fusion protein can be expressed in cis or trans andused in various states of purity.

The curli system of Escherichia coli is composed of small proteinmonomers, CsgA, that are secreted by the cell and self-assembleextracellularly into highly robust amyloid nanofibers that are anchoredto the cell surface by an outer-membrane bound homologous protein,CsgB.⁵ The resulting curli nanofibers have a diameter of ˜7 nm, form atangled curly mass, and are resistant to boiling in detergent.Incubation of curli fibers in ˜90% formic acid is required to dissociatethe amyloid nanofiber into its monomers. We have demonstrated that it ispossible to make genetic fusions to the CsgA protein while maintainingits ability to form extracellular curli fibers (FIG. 1A). This wasaccomplished by creating a panel of mutants (schematically shown in FIG.1B, left) consisting of CsgA fused at the N- or C-terminus by variousflexible linkers to a metal binding domain (MBD), a peptide domain fromthe Pseudomonas spp. known to bind strongly to stainless steelsurfaces.⁶ The csgA variants were cloned into plasmids and transformedinto a strain of E. coli (LSR10) missing the wild-type csgA gene butcontaining the remaining curli processing machinery. Therefore, uponinduction, amyloid formation could be attributed solely to theheterologously engineered CsgA fusion mutants. Congo Red (CR) stainingof bacterial colonies on low-salt media is a standard colorimetricindicator for amyloid fibril formation, in which a red coloration of thebacteria indicates successful Curli fiber formation. The results of theinsertion panel show that only the C3 fusion, which has the longestlinker between the CsgA C-terminus and the MBD, is able to form anappreciable amount of amyloid fibers.

To test the extent of the functional domains that can be fused to theC-terminus of CsgA, a library of different functional peptides andprotein domains ranging in size from 7 to 59 residues was selected andcloned into the C-terminal region, while retaining the flexible linker(FIG. 1C). FIG. 1D of the curli fusion constructs streaked onto CRplates indicates that various small functional peptides are tolerated bythe curli machinery and form curli nanofibers as evidenced by CRstaining, but the 59-amino acid Mms6 domain does not exhibitamyloid-positive CR staining. TEM and FE-SEM images visualizing thecurli fibers that result from some of the CsgA fusion proteins supportthis data, with the MBD and SpyTag peptide fusions at the C3 insertionsite producing visible nanofibers (FIGS. 1E-1F). These resultsdemonstrate that 1) it is possible to make genetic fusions to CsgA whilemaintaining processing by the cellular curli machinery for secretionfrom the cell and assembly into amyloid fibers extracellularly, and 2) AC-terminal insertion site with a flexible linker is the most tolerantconstruct architecture for curli secretion and assembly.

Curli-Displayed Fusion Peptide Domains are Functional and can be Used asa Scaffold for the Presentation of Proteins.

To test if the peptide domains are functional when displayed on theextracellularly assembled engineered curli nanofibers, a peptide tagthat specifically interacts with a protein domain was expressed in theengineered curli system (FIG. 2A). This system is a recentlydemonstrated a covalent capture platform in which the protein domain(called “SpyCatcher”, herein referred to as “SC”.) is able tospecifically and robustly form an isopeptide bond with the peptide tag(called “SpyTag”, herein referred to as “ST”.).⁷ Thus a functionallydisplayed ST peptide on the curli biofilm will form an irreversible bondto exogenously added SC protein domain that can be fused to any targetprotein. A reporter fusion protein of a fluorescent protein (Venus) tothe SC domain was designed, expressed, and purified. When this purifiedVenus-SC protein was added to the wild-type curli biofilm displaying nopeptide, no localization of fluorescence to the biofilm was observed(FIG. 2B, Row 1). However, when bioengineered curli biofilm displayingthe ST peptide was exposed to the Venus-SC protein, significantfluorescence localized to the biofilm was observed (FIG. 2B, Row 2). Incontrast, a Venus-SC(E77Q) mutant that is unable to catalyze thecovalent bond to the ST peptide does not display strong localizedfluorescence (FIG. 2B, Row 3). These results demonstrate: 1) peptidesdisplayed on curli nanofibers are expressed in a functional andaccessible form, and 2) that target proteins can be immobilized onpeptide-functionalized curli biofilms by fusing the target protein to aprotein domain that interacts with a peptide which is displayed by ourengineered curli biofilm technology.

The purification of expressed proteins is not requisite for the proteinimmobilization strategy. Rather, like many of the affinity purificationtechnologies currently used (i.e., chitin binding or Ni-NTA beads), thecurli-SpyTag system can capture SpyCatcher-fusion proteins from celllysate without extensive purification. In our experiments, Venus-SC andVenus-SC(E77Q) were expressed, cells lysed to release the proteins, thecell debris pelleted and the clarified cellular lysate added directly tothe biofilms. As in the experiments described above utilizing purifiedprotein, the Venus-SC is not captured by biofilms expressing curliwithout ST (FIG. 2C(i)), but is captured by curli-ST expressing biofilms(FIG. 2C(ii)). Again, the Venus-SC(E77Q) mutant is not captured by thesecurli-ST biofilms (FIG. 2C(iii)). This contributes to the robustness ofthe engineered curli platform as it combines purification and functionalmaterial synthesis into one step: the bacteria produces both the biofilmscaffold and the target protein to be immobilized. Applications as adisruptive technology for industrial biotransformation processes arecontemplated, in which multiple complex bioreactor steps can beintegrated into a single genetically programmed culture. This reducesthe overall cost and increases the efficiency of system setup, which isa major concern for industry adaption.⁸

The protein immobilization onto biofilm using the curli peptide displaytechnology may use any of the various technologies consisting of aninteracting peptide tag and protein domain, including but not limitedto: the SpyTag-SpyCatcher system,⁷ the BCCP-Biotin Ligase-Streptavidinsystem,⁹ or Sortase-mediated Ligation.¹⁰ The technology described hereinis broadly applicable for the biofilm-immobilization of enzymatic,optically active, electronically active, biotemplating, structural,stimulus-responsive, and binding proteins or any combination thereof.

Applications

The curli system described herein is a biologically producedpeptide-functionalized surface coating capable of being programmed tospecifically immobilize another chemical or biological entity or toexhibit specific binding properties. The displayed peptide may possessintrinsic properties such as binding to other exogenously addedfunctional components, such as inorganic nanoparticles (especially thosewith interesting opto-electronic properties or magneto-responsiveness),carbon-based nanostructures (i.e., graphene or nanotubes, which mayconfer conductivity), or environmental toxins (i.e., hormones or toxicmetals). The engineered biofilms can also be used to display peptidesthat template the formation of inorganic or organic materials.Functionalizing the biofilm with peptides that specifically bind todifferent materials allows the surface coating of these materials in agenetically programmable manner. In addition, applications whereby theliving biofilm is used to immobilize and present any arbitrary protein,as might be useful for applications in biocatalysis, biotemplating, orbiosensing are specifically contemplated. In contrast to otherengineered systems that serve the same purpose, the synthesis andassembly of the material described herein is accomplished entirely bythe bacterial cell, which acts as a factory for the production ofprogrammed nanomaterials.

Potential specific applications include:

Biologically-produced nanomaterials that have programmable optical,magnetoresistive or semiconductor properties from either thepeptide/immobilized protein itself or by the induction of templatedmaterials.

By displaying catalytic peptides or enzymes on the curli biofilm, asystem for high-efficiency immobilized biocatalysis in which variousimmobilization substrates can be used for the adhesion of the biofilmand which can be used in any bioreactor design is contemplated.

The peptide/immobilized proteins can also encode for biologically activebiomolecules that will allow the biofilm to act as a tissue scaffold orvaccine delivery material.

Expression of peptide/immobilized proteins that bind to or enzymaticallyneutralize environmental toxins such as synthetic hormones, smallmolecules, or toxic metals can be used as a biofilm-based technology forbioremediation.

By expressing peptides that are able to specifically bind to preciousmetals such as gold, silver, platinum, and rhodium on the biofilmsdescribed herein, there is a vast possible active surface area for theprofitable recovery of such precious materials.

The curli nanofibers can be engineered as conductive nanowires fornumerous advanced materials applications by the display ofpeptides/proteins that are inherently conductive, or by thetemplating/anchoring of materials that are conductive.

The use of bacteria to generate nanowires for energy storage based uponthe expression on the curli biofilm of peptides capable of templatingconductive or semiconductive materials, such as FePO4.

Bacteria can be specifically engineered via the displayed peptide tobind strongly to specific substrates, such as steel, glass, or gold.Such material-specific binding can form the foundation of abiofilm-based biosensing technology.

The curli nanofiber matrix can also be engineered to displaypeptides/proteins that interact with other molecules in order to enhanceor alter the mechanical properties of another material.

By engineering the curli to adhere to specific materials, the biofilmcan act as a living coating capable of providing adaptive andregenerative benefits, such as biocatalysis on a wide variety ofimmobilization substrates, corrosion resistance to the material,enhanced biofilm coverage for microbial fuel cell applications, or actas an environmentally responsive organic(biofilm)-inorganic(substrate)material.

Discussion

Biofilms are used on large scales in technologies for bioremediation andwaste water treatment.¹¹⁻¹³ The use of biofilms for still furtherapplications, such as microbial fuel cells,¹⁴⁻¹⁹biocatalysis,^(8, 20-24) and corrosion prevention.²⁵⁻²⁷ has beeninvestigated. However, these applications rely on the intrinsiccapabilities of naturally occurring microbes. For example, biofilms usedin the context of removing heavy metals from water samples employ soilbacteria that are known to have the capacity to sequester metal ions;microbes used in fuel cells are most often those that are known tonaturally produce electroconductive extracellular components. Onesubstantial drawback to these existing methods is that it requires acell that is capable of internalizing the substrate to be bound orbiocatalyzed. This severely limits the efficiency of the process byadding a kinetic diffusion barrier. By contrast, the technologydescribes herein provides a bacterial biofilm component (curli) whosecapabilities have been enhanced or augmented with completely new onesbased on a rational genetic engineering approach.

Although this circumvents barriers of substrate accessibility mentionedabove, The curli-based peptide display system has a number of distinctbenefits over traditional cell surface display systems²⁸⁻³¹. In thetechnology described herein, each cell acts as a factory to generateengineered nanofibers, and therefore greatly increases the functionalsurface area of the scaffold available for the display of the peptides.In addition, the curli fibers are extremely stable and can exist evenafter the removal of the cells, whereas prior cell-surface displaysystems are vulnerable to harsh conditions which may cause lysis of thecells.

The technology described herein provides a programmable functionalizedbiofilm, including the immobilization of proteins, which is an advanceover the prior art and greatly expands the potential applications ofthis platform technology. Further, the present technology is preferableto fimbrial display in that curli nanofibers form through theself-assembly of a single monomeric protein, CsgA, whereas the fimbriaesystems The fact that the curli forms the major protein component of E.coli biofilms and is a simple genetic system composed of a singleprotein monomer provides distinct advantages to the present describedengineered curli platform.

The technology described herein is contemplated to have a number ofapplications, including but not limited to, biocatalysis, metalrecovery, and electrobiological applications. However, the engineeredbiofilms can be useful for any applications where surface coatings withprogrammable functions are required. In addition to acting as a materialitself, the curli-based system can be useful as a screening tool torapidly identify or even evolve self-assembling proteins with desiredbehaviors. The extracellular nature of the curli biofilm, its intrinsichigh stability, and vast potential for a high-surface area material willmake this technology highly valuable in various biocatalysis,bioremediation, and biomedicine applications.

REFERENCES

-   1. Giltner, C. L. et al. The Pseudomonas aeruginosa type IV pilin    receptor binding domain functions as an adhesin for both biotic and    abiotic surfaces. Molecular Microbiology 59, 1083-1096 (2006).-   2. Duguid, J. P., Anderson, E. S. & Campbell, I. Fimbriae and    adhesive properties in Salmonellae. The Journal of pathology and    bacteriology 92, 107-138 (1966).-   3. Hidalgo, G., Chen, X., Hay, A. G. & Lion, L. W. Curli Produced by    Escherichia coli PHL628 Provide Protection from Hg(II). Applied and    Environmental Microbiology 76, 6939-6941 (2010).-   4. Hall-Stoodley, L., Costerton, J. W. & Stoodley, P. Bacterial    biofilms: from the natural environment to infectious diseases. Nat    Rev Microbiol 2, 95-108 (2004).-   5. Hammer, N. D., Schmidt, J. C. & Chapman, M. R. The curli    nucleator protein, CsgB, contains an amyloidogenic domain that    directs CsgA polymerization. Proceedings of the National Academy of    Sciences of the United States of America 104, 12494 (2007).-   6. Davis, E. M., Li, D.-y. & Irvin, R. T. A peptide—stainless steel    reaction that yields a new bioorganic—metal state of matter.    Biomaterials 32, 5311-5319 (2011).-   7. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a    protein, through engineering a bacterial adhesin. Proceedings of the    National Academy of Sciences 109, E690-7 (2012).-   8. Rosche, B., Li, X. Z., Hauer, B., Schmid, A. & Buehler, K.    Microbial biofilms: a concept for industrial catalysis? Trends in    Biotechnology 27, 636-643 (2009).-   9. Beckett, D., Kovaleva, E. & Schatz, P. J. A minimal peptide    substrate in biotin holoenzyme synthetase-catalyzed biotinylation.    Protein science: a publication of the Protein Society 8, 921-929    (1999).-   10. Mao, H., Hart, S. A., Schink, A. & Pollok, B. A.    Sortase-Mediated Protein Ligation: A New Method for Protein    Engineering. Journal of the American Chemical Society 126, 2670-2671    (2004).-   11. McNamara, C. J., Anastasiou, C. C., O'Flaherty, V. &    Mitchell, R. Bioremediation of olive mill wastewater. International    Biodeterioration &amp; Biodegradation 61, 127¬134 (2008).-   12. Valls, M. & De Lorenzo, V. Exploiting the genetic and    biochemical capacities of bacteria for the remediation of heavy    metal pollution. FEMS microbiology reviews 26, 327-338 (2002).-   13. Wang, Y.-K. et al. Development of a Novel Bioelectrochemical    Membrane Reactor for Wastewater Treatment. Environmental Science    &amp; Technology 45, 9256-9261 (2011).-   14. Erable, B., Duteanu, N. M., Ghangrekar, M. M., Dumas, C. &    Scott, K. Application of electro-active biofilms. Biofouling 26,    57-71 (2010).-   15. MANSFELD, F. The interaction of bacteria and metal surfaces.    Electrochimica Acta 52, 7670-7680 (2007).-   16. Strycharz-Glaven, S. M., Snider, R. M., Guiseppi-Elie, A. &    Tender, L. M. On the electrical conductivity of microbial nanowires    and biofilms. Energy &amp; Environmental Science 4, 4366 (2011).-   17. Wang, Z.-W. & Chen, S. Potential of biofilm-based biofuel    production. Applied Microbiology and Biotechnology 83, 1-18 (2009).-   18. Yu, Y.-Y., Chen, H.-1., Yong, Y.-C., Kim, D.-H. & Song, H.    Conductive artificial biofilm dramatically enhances bioelectricity    production in Shewanella-inoculated microbial fuel cells. Chemical    Communications 47, 12825 (2011).-   19. Nevin, K. P., Woodard, T. L., Franks, A. E., Summers, Z. M. &    Lovley, D. R. Microbial Electrosynthesis: Feeding Microbes    Electricity To Convert Carbon Dioxide and Water to Multicarbon    Extracellular Organic Compounds. mBio 1, e00103-10-e00103¬10 (2010).-   20. Li, X. Z., Hauer, B. & Rosche, B. Single-species microbial    biofilm screening for industrial applications. Applied Microbiology    and Biotechnology 76, 1255-1262 (2007).-   21. Li, X. Z., Webb, J. S., Kjelleberg, S. & Rosche, B. Enhanced    benzaldehyde tolerance in Zymomonas mobilis biofilms and the    potential of biofilm applications in fine-chemical production.    Applied and Environmental Microbiology 72, 1639-1644 (2006).-   22. Gross, R., Hauer, B., Otto, K. & Schmid, A. Microbial biofilms:    New catalysts for maximizing productivity of long-term    biotransformations. Biotechnology and Bioengineering 98, 1123-1134    (2007).-   23. Tsoligkas, A. N. et al. Engineering Biofilms for Biocatalysis.    Chem Bio Chem 12, 1391-1395 (2011).-   24. Wood, T. K., Hong, S. H. & Ma, Q. Engineering biofilm formation    and dispersal. Trends in Biotechnology 29, 87-94 (2011).-   25. Zuo, R. Biofilms: strategies for metal corrosion inhibition    employing microorganisms. Applied Microbiology and Biotechnology 76,    1245-1253 (2007).-   26. Stadler, R. et al. First evaluation of the applicability of    microbial extracellular polymeric substances for corrosion    protection of metal substrates. Electrochimica Acta 54, 91-99    (2008).-   27. Jayaraman, A., Sun, A. & Wood, T. Characterization of axenic    Pseudomonas fragi and Escherichia coli biofilms that inhibit    corrosion of SAE 1018 steel. Journal of applied microbiology 84,    485-492 (2011).-   28. Li, D., Newton, S. M. C., Klebba, P. E. & Mao, C. Flagellar    Display of Bone-Protein-Derived Peptides for Studying    Peptide-Mediated Biomineralization. Langmuir: the ACS journal of    surfaces and colloids 28, 16338-16346 (2012).-   29. van Bloois, E., Winter, R. T., Kolmar, H. & Fraaije, M. W.    Decorating microbes: surface display of proteins on Escherichia    coli. Trends in Biotechnology 29, 79-86 (2011).-   30. Wang, A. A., Mulchandani, A. & Chen, W. Whole-Cell    Immobilization Using Cell Surface-Exposed Cellulose-Binding Domain.    Biotechnology progress 17, 407-411 (2001).-   31. Georgiou, G. et al. Display of heterologous proteins on the    surface of microorganisms: from the screening of combinatorial    libraries to live recombinant vaccines. Nature Biotechnology 15,    29-34 (1997).-   32. Chapman, M. R. et al. Role of Escherichia coli curli operons in    directing amyloid fiber formation. Science 295, 851-5 (2002).-   33. Der Vartanian, M. et al. An Escherichia coli CS31A fibrillum    chimera capable of inducing memory antibodies in outbred mice    following booster immunization with the entero-pathogenic    coronavirus transmissible gastroenteritis virus. Vaccine 15, 11120    (1997).-   34. Der Vartanian, M. et al. Permissible peptide insertions    surrounding the signal peptide-mature protein junction of the ClpG    prepilin: CS31A fimbriae of Escherichia coli as carriers of foreign    sequences. Gene 148, 23-32 (1994).-   35. Méchin, M. C., Der Vartanian, M. & Martin, C. The major subunit    ClpG of Escherichia coli CS31A fibrillae as an expression vector for    different combinations of two TGEV coronavirus epitopes. Gene 179,    211-8 (1996).-   36. White, A. P., Collinson, S. K., Banser, P. A., Dolhaine, D. J. &    Kay, W. W. Salmonella enteritidis fimbriae displaying a heterologous    epitope reveal a uniquely flexible structure and assembly mechanism.    J Mol Biol 296, 361-72 (2000).-   37. White, A. P. et al. High efficiency gene replacement in    Salmonella enteritidis: chimeric fimbrins containing a T-cell    epitope from Leishmania major. Vaccine 17, 2150-61 (1999)

Example 2

Because of their role in bacterial pathogenicity and persistence, thevast majority of research on biofilms has focused on preventing theirformation and promoting their dispersal. However, this has resulted inan overlooked opportunity to develop biofilms as functional materials.Demonstrated herein is a new technology platform, Biofilm-IntegratedNanofiber Display (BIND), in which the display of functional peptides isgenetically engineered on the curli system of E. coli, the majorproteinaceous component of the biofilm matrix consisting of thinamyloidogenic nanofibers. Bacteria in such a system are contemplated asserving as a living foundry for the production, assembly, andpost-processing of customized advanced biomaterials and programmablesurface coatings. Herein, a fusion site in the structural curli genecsgA is identified that allows for the display of peptides whileretaining the ability of secreted CsgA chimeras to self-assemble intocurli fiber networks. A library of functional peptides with a range ofsizes and secondary structures were recombinantly engineered into thecurli biofilms, demonstrating the modularity and flexibility of thecurli display system. Furthermore, the kinetics and stability of theBIND nanofibers are established and it is demonstrated that the peptidesare fully displayed, as designed. Finally, the retention of peptidefunctionality in BIND biofilms was demonstrated in three broadapplications: engineered adhesion, peptide catalysis, and proteinimmobilization.

Introduction

In nature, most bacteria exist as biofilm communities, residing in aself-generated protective nanoscale scaffold of proteins, sugars,lipids, and extracellular DNA that defends against environmentalrigors⁵. Biofilm formation is essential for bacterial adhesion andcolonization of both natural and man-made surfaces. Characterization ofbiofilms in the mid-20^(th) century revealed their key role in microbialpersistence and pathogenicity, which has recently led to an abundance ofresearch on biofilm prevention and dispersal.^(6, 7) However, thesehighly evolved extracellular matrices hold untapped potential as abeneficial nanobiotechnology engineering platform. There is asignificant body of work that investigates the use of biofilms forbeneficial purposes such as wastewater treatment⁸⁻¹⁰ andbiotransformations¹¹⁻¹³, but these efforts focus on the use of naturallyoccurring organisms that happen to have evolved various desiredqualities. Efforts to rationally engineer the structure of biofilms atthe molecular level have, to our knowledge, been completely absent. Todate, there exists no robust and broad technology for the facileengineering of biofilm components.

The presently described approach to controlling the molecularcomposition of biofilm-based materials relies on the curli system—aproteinaceous component of some E. coli biofilms. The curli system iscomposed of a small 13-kDa-protein monomer, CsgA, that is secreted bythe cell and self-assemble into highly robust amyloid nanofibers thatare anchored to the cell surface by a homologous outer-membrane protein,CsgB¹⁴⁻¹⁶. The resulting curli nanofibers have a diameter of ˜7 nm andform a tangled curly mass that encapsulates the cells. Curlibiosynthesis is promoted by an operon that contains seven genes(csgA-G).¹⁸ Of these, CsgA is the main structural component,¹⁹ while theother proteins are involved in the nucleation of amyloid fibers(CsgB),²⁰ or the processing (CsgE, F),21, ²² secretion (CsgC, G)23, ²⁴,and control of transcription (CsgD)¹⁷ of CsgA. The curli system wasselected for this technology because it exhibits several features thatmake it amenable to the type of materials engineering platform which iscontemplated herein. First, the amyloid fibers formed by CsgA areextremely robust, being able to withstand boiling in SDS²⁵, increasingtheir potential utility in harsh environments. Second, since theextracellular fiber network is composed primarily of a single protein,its structural features can be easily controlled by manipulating asingle gene. Finally, although analogous extracellular amyloid systemsexist in other organisms, notably Salmonella26, ²⁷ and Pseudomonas, ²⁸the curli system is by far the best studied, and the fact that it occursnatively in E. coli and consists of a single structural protein makes ithighly genetically tractable. Although some of these other fimbriaesystems have been investigated as potential vaccine delivery agents,²⁹there has been no research into their use in other biofilm-basedbiotechnological applications.

Described herein is a strategy that referred to as “biofilm-integratednanofiber display” (BIND), which allows the programming of an E. colibiofilm's functional properties by genetically appending functionalpeptide domains to the CsgA protein. After the new CsgA-peptide issecreted and assembled, the amyloid nanofiber network displays thepeptide in very high density on its surface. The biofilm's function isthen augmented according to the sequence of the displayed peptides. Itis demonstrated herein that functional peptide domains of variouslengths and secondary structures can be appended to CsgA withoutprecluding the formation of curli fibers. Furthermore, the effect ofpeptide domain fusion on the self-assembly kinetics of the CsgA mutantsis quantified. Lastly, it is demonstrated that the peptide domainsmaintain their function in the context of the biofilm after secretionand assembly.

Results

Design of BIND for Programmable Functionalized Biofilms

For the design of the BIND platform described herein, a number ofconsiderations were taken into account. The system has to be geneticallytractable and modular, allowing for the facile integration of anyfunctional peptide domain into the biofilm. This precludes the use ofthe polysaccharide biopolymers that form the bulk of biofilmmass,^(30, 31) as their synthesis relies on multi-enzymatic pathwaysthat are difficult to engineer.³² Proteinaceous components of biofilmsknown as fimbriae, which form cell-anchored nanoscale protein fibers,were selected as the scaffold of choice. Of the fimbriae systems inbacteria, the curli system was chosen as these amyloid nanofibers areprimarily composed of a single self-assembling protein, CsgA. Thismaximizes the representation of the functional domain in the assemblednetwork and greatly simplifies the complexity of the system.Furthermore, this system is native to E. coli, providing a wealth ofgenetic tools and expression technologies to work with. Therefore, itwas first decided to test fusions of the functional domain to the N- orC-terminus of CsgA.

C-Terminal Peptide Fusions to CsgA are Able to Form Curli Nanofibers

The goal was to create genetic fusions to the CsgA protein that maintainits ability to form curli fibers. This was accomplished by creating apanel of mutants (schematically shown in FIG. 3A) consisting of CsgAfused at the N- or C-terminus to a metal binding domain (MBD), a peptidedomain from the Pseudomonas spp. known to bind strongly to stainlesssteel surfaces.³³ Three variants were prepared for each terminus: MBD islinked to CsgA either directly, or with a short (GS) or long (GSGGSG)flanking linker. The csgA variants were cloned into plasmids andtransformed into a strain of E. coli (LSR10) missing the wild-type csgAgene, but containing the remaining curli processing machinery.³⁴Therefore, upon induction, amyloid formation could be attributed solelyto the heterologously engineered CsgA fusion mutants. As a readout ofcurli fiber production, Congo Red (CR) staining of bacterial colonies onlow-salt media was used, which is a standard colorimetric indicator foramyloid fibril formation.¹⁴ The results of the insertion panel show thatonly the C3 fusion, which has the longest linker between the CsgAC-terminus and the MBD, is able to form an appreciable amount of amyloidfibers (FIG. 1B). Other fusions are not tolerated, as evidenced by thelack of CR staining (FIG. 1B). TEM images visualizing the curli fibersthat result from the CsgA fusion proteins support this data, with the C3insertion site producing visible nanofibers (data not shown). Theseresults clearly demonstrate that it is possible to make C-terminalgenetic fusions to CsgA without inhibiting its secretion from the celland extracellular assembly into amyloid fibers.

The C3 Design Allows for Modular Incorporation of Various Peptides intoCurli Biofilms.

To test the modularity of the functional domains that can be fused tothe C-terminus of CsgA, a library of peptide domain fusions ranging insize from 7 to 59 amino acids while maintaining the 6 amino acidflexible linker from C3 was created (Table 1). The library alsorepresents different secondary structures, as most of the peptides arenot designed to exhibit any defined conformation, while MBD and Mms635contain intramolecular disulfide bonds, which should lock them into amore rigid conformation. Finally, the library members were designed tospan a range of functions that might be potentially useful in futureapplications of the BIND system to various technologies, includingcapturing proteins³⁶ and binding to inorganic nanoparticles³⁷⁻³⁹ andsurfaces.³³ The library members were cloned into LSR10 cells and probedfor the formation of curli-based amyloid networks by CR stainingPositive CR staining (FIG. 2A) and quantitative analyses (FIG. 4) formost of the library members suggests that small peptide fusions weretolerated by the curli export machinery and successfully assembled intoextracellular amyloid networks. The only mutant for which there was nopositive staining was the 59-amino acid Mms6 domain. CsgA is thought tobe transported across the outer membrane by the CsgG complex as anunfolded conformer¹⁵. Without wishing to be limited by theory, giventhat the pore size of the CsgG complex is estimated to be ˜2 nm,²⁴ thissuggests that larger folded domains may not be compatible with the curliexport machinery.

TEM imaging of the modified curli biofilms suggests that theCsgA-peptide fusions assemble into nano-scale fibers similar to thoseobserved for wt-CsgA (data not shown). The fibers display acharacteristic tangled curly morphology and appear to be closelyassociated with the cell surface. The TEM images were intentionallyobtained with diluted samples so that the nanostructure of the fiberscould be easily discerned. The fibers in these images that appear to befully extended are likely an artifact of the drying process duringsample preparation and do not represent the native fiber morphology.Furthermore, SEM imaging shows that the modified curli biofilms can bevery dense and several cell layers thick (data not shown) whilemaintaining a highly interconnected network of fibers between cells.

In Vitro Self-Assembly Kinetics of CsgA-Peptide Fusions.

In order to determine the effect of peptide domain fusion on theself-assembly of CsgA, several variants were selected for purificationand assembly studies in vitro. For purification purposes, theCsgA-peptide fusions were appended to a His-tag followed by anenterokinase cleavage sequence. The purification sequences were insertedin place of the Sec tag such that the proteins would not be exported tothe periplasm and after affinity purification from cell lysates, theenterokinase cleavage yielded proteins that were identical to thosesecreted by the corresponding LSR10 transformants after processing ofthe native Sec tag⁴⁰. The purified proteins can monitored for theirassembly kinetics using an established thioflavin T (ThT) assay.

Functionality of Peptides Displayed with BIND.

The BIND system is capable of introducing a variety of novel functionsto curli-based biofilms. Therefore, in addition to confirming secretionand assembly of CsgA-peptide chimeras, it was also sought to demonstratethat the fused peptide domains maintain their cognate functions in thecontext of the fully formed biofilms. Accordingly, two peptides wereselected from Table 1 (MBD and SpyTag) and their ability to augmentbiofilm performance was tested. MBD was chosen because its affinity forsteel should enhance the adhesion of curli-based biofilms to stainlesssteel surfaces. To test this hypothesis, LSR10 cells expressing theCsgA-MBD mutant were grown in culture and, after induction, spotted ontostainless steel 304L coupons and allowed to dry in air. The sameprocedure was followed with cells expressing wt-CsgA and no CsgA asnegative controls. Each coupon included an array of three spots—one fromeach culture. The coupons were then subjected to vigorous washing bysubmerging them in aqueous buffer and vortexing (FIG. 5A). Biofilmscomposed of the CsgA-MBD fusion clearly withstood the washing procedure,while those expressing wt-CsgA or no CsgA were easily washed off thesurface (FIGS. 5B-5D). Based on this data, it is concluded that theadhesion of curli-based biofilms to non-natural surfaces can beartificially enhanced by appending peptide domains that have beenpre-selected to exhibit a desired function.

As a second demonstration of the utility of the BIND system, theCsgA-SpyTag mutant was investigated as a means to immobilize full-lengthproteins to the curli matrix. The SpyTag-SpyCatcher system is a recentlydeveloped strategy for protein capture that uses a CnaB2 protein thathas been split into a 13 amino acid peptide (SpyTag) and a 15-kDaprotein (SpyCatcher).³⁶ When brought together, the two fragmentscatalyze the formation of an intermolecular isopeptide bond. It wassought to use this strategy to circumvent the apparent size limitationsof the curli export machinery by enabling covalent bond formationbetween the curli network and larger proteins using completelygenetically encodable components. Accordingly, biofilms displayingcontaining the CsgA-SpyTag chimera were formed on a surface-modifiedglass substrate using PHL628 cells, an E. coli strain that has beenengineered to overproduce CsgA. A SpyCatcher-Venus fusion protein wasused to probe for the presence and functionality of the SpyTag domain.Venus is a green fluorescent protein variant with enhanced opticalproperties. The glass-immobilized biofilms were treated with a nucleicacid stain (SYTO 61, Invitrogen) to test for the presence of cells.Subsequently, they were treated with a solution containing eitherSpyCather-Venus, or SpyCatcherEQ-Venus, a mutant in which the covalentbond formation has been abolished. Following extensive washing steps,the biofilms were imaged using fluorescence microscopy. Only the propercombination of biofilms expressing the CsgA-SpyTag and treatment withSpyCatcher-Venus resulted in significant co-localization of the twofluorescent signals (FIG. 6F). Biofilms expressing wt-CsgA were unableto capture the fluorescent protein. Similarly, biofilms expressingCsgA-SpyTag did not exhibit green fluorescence when treated with thenon-functional SpyCatcherEQ-Venus mutant. Together these results suggestthat SpyTag peptide can be fused to CsgA and maintain its functionalityafter formation of the curli network

Discussion

The straightforward self-assembling system presented herein allows forthe precise molecular control of bacterial extracellular matrixcomposition by genetic engineering and establishes a platform for thecreation of functional bionanomaterials from living systems and perhapseven living functional materials. The advantages of such a syntheticbiology platform are numerous: curli fibers can be engineered to displaya variety of peptides with useful features, such as binding to orbiotemplating the synthesis of inorganic materials, enhancing biofilmadhesion to particular surfaces, or providing a scaffold-like surfacecoating for the immobilization of other biomolecules. The biofilm itselfis a “green” (i.e., environmentally friendly) material in that it ismade biosynthetically and requires no petroleum-derived raw buildingblocks. Additionally, the biofilm has the capacity to be aself-generating and self-repairing renewable material.

Surface modification and functionalization is ubiquitous in nearly allaspects of our society. However, the use of biologically-derived surfacecoatings are lacking. Life has evolved a highly efficient coatingstrategy that was an early evolutionary adaptation enabling thebacterial colonization of surfaces⁴¹. Current applications of biofilmsfor applied technology utilize naturally occurring biofilms orco-cultures of biofilm-forming bacteria⁴² to generate the desiredfunctionality for thin-film biocatalysis or bioremediation. Theseapplications remain limited and the adoption of biofilm-based technologyin other industries is greatly hindered by an inability to programbiofilm functionality and control the temporal dynamics of biofilmformation.

The curli system of E. coli plays a central role in host-cell adhesionof enteropathogenic strains and is critical for the formation ofbiofilms²⁵ Curli has been extensively studied as a model system forfunctional bacterial amyloids¹⁸ as well as for the development ofbiofilm-inhibiting compounds⁴³. The curli system is demonstrated hereinto be ideal as a programmable biofilm platform as it is predominantlycomposed of a single genetically programmable unit, the self-assemblingCsgA proteins.

Materials and Methods

Cell Strains and Plasmids.

All cloning and protein expression was performed in Mach1™ (Invitrogen)and Rosetta™ cells (EMD), respectively. The csgA gene was isolated fromE. coli K-12 genomic DNA and cloned into pBbE1a, a ColE1 plasmid undercontrol of the Trc promoter⁴⁴. Expression vectors were constructed usingpET30a plasmids (EMD), with the native N22 region of the CsgA proteincloned immediately after the enterokinase cleavage site. Peptide insertregions were either fully synthesized (Integrated Dna Technologies) orPCR-generated by overlap extension. All cloning was performed by usingisothermal Gibson Assembly as described⁴⁵ and verified by DNAsequencing.

Curli Biofilm Formation

To produce curli, LSR10 cells or PHL628 cells were transformed withpBbE1a plasmids encoding for CsgA or CsgA-peptide fusions. As a negativecontrol, cells were transformed with empty pBbE1a plasmid. The cellswere then streaked or spotted onto YESCA-CR plates, containing 10 g/L ofcasamino acids, 1 g/L of yeast extract, and 20 g/L of agar. All mediacomponents were from Fisher. The plates were supplemented with 100 mg/mLof ampicillin, 0.5 mM of IPTG, 25 mg/mL of Congo Red and 5 mg/mL ofBrilliant Blue G250. The plates were then incubated for 48 hours at 25°C. and then imaged to determine the extent of Congo Red binding. For thespotted plates, the transformants were grown in YESCA liquid mediasupplemented with 100 mg/mL of ampicillin and 0.2 mM of IPTG for 48hours at 25° C. before spotting 20 mL onto YESCA-CR plates. This sameYESCA liquid induction procedure was used to prepare samples for CsgApurification, and electron microscopy.

Quantitative Congo Red Binding Assays

Determination of Congo Red binding was adapted from previously publishedmethods. Briefly, transformant cultures grown on YESCA plates for 48hours at 25° C. were scraped and resuspended gently in PBS. The cellresuspention was adjusted to an OD600 of 3. To 1 mL of this, a Congo Redsolution was added to a final concentration of 0.001% and allowed toincubate at 4° C. for 1 hour. The cells were then pelleted and the 490nm absorbance of 200 μL of the supernatant was measured in a BIOTEK H1microplate reader. The amount of Congo Red binding was determined as thesubtractive amount of this measurement against a PBS+Congo Red control.All samples were performed in triplicate.

Chimeric CsgA Purification

Rosetta™ cell transformants were grown in LB until mid-log phase andinduced with 0.2 mM IPTG for 3 hours. The cells were pelleted and thenfrozen at −20° C. for subsequent purification. The pellets were thawedand lysed in BugBuster Protein Extraction Reagent (EMD), 1 mg/mLLysozyme, 50 μg/mL DNase, and protease inhibitors (Roche). After 30minutes, the lysate was diluted into a solution of 8 guanidinehydrochloride, 250 mM NaCl, and 50 mM Tris at a pH of 7.5 and incubatedfor 16 hours to dissolve aggregates. Any insoluble mass was pelleted bycentrifugation at 18,000 rpm for 30 minutes, the clarified lysate wasfiltered through a 0.22 micron filter, and then incubated with Ni-NTAresin (Qiagen) for 2 hours. The protein-bound resin was then washed with8 guanidine hydrochloride, 250 mM NaCl, 0.1% Triton X-100, 1 mM DTT, and50 mM Tris (pH of 7.5) and eluted with the same buffer supplemented with200 mM imidiazole. The eluate was dialyzed into EK cleavage buffer (1MUrea, 20 mM methylamine, 50 mM Tris, pH 7.5) and then incubated with 3ng of enterokinase (Roche) for 24 hours. The cleaved CsgA protein wasthen lyophilized, treated with 100 μL of HFIP to dissolve any curlifibers, and stored as a dried powder.

ThT Kinetic Assay

Immediately before the ThT assay, the cleaved, HFIP-treated protein wasresolubilized into 8M guanidine hydrochloride, 250 mM NaCl, 0.1% TritonX-100, and 50 mM Tris at a pH of 7.5. This solution was FPLC purified ona Sephadex-G75 gel-filtration column to remove dimers and oligomers. Thefraction containing the monomeric CsgA fusions were then desalted andthe concentration determined by UV absorbance. The ThT assay wasimmediately performed with 30 μM of the CsgA fusion or wild-type proteinwith 40 μM ThT; the fluorescence was measured in a SpectramaxM2 platereader at 438ex/495em.

TEM and SEM

Curliated wildtype or BIND cell samples were either directly taken frominduced YESCA cultures or scraped from YESCA-CR plates and resuspendedin Millipore H2O. For TEM analysis, 5 mL of the sample was spotted ontoformvar-carbon grids (Electron Microscopy Sciences), washed twice withMillipore H2O, and stained for 15 seconds with 1% uranyl formate beforeanalysis on a JEOL 1200 TEM. For SEM analysis, samples were applied toNucleopore filters under vacuum, washed with Millipore H₂O and fixedwith 2% glutaraldehyde+2% paraformaldehyde overnight at 4° C. Thesamples were then washed in Millipore H2O, dehydrated with an increasingethanol step gradient, and dried using an hexamethyldisilazane stepgradient before gold sputtering and analysis on a Zeiss Supra 55VP™FE-SEM.

Immunogold TEM

For anti-FLAG immunogold labeling of the BIND cells displaying the FLAGtag, the cells were first adhered to the TEM grid as described above.Then, the grids were washed 3× in blocking buffer (PBS+1% BSA), floatedon a drop containing a 1:1000 dilution of primary anti-FLAG murineantibody in PBS for XX minutes, washed in blocking buffer again, andthen floated on a drop of 1:1000 diluted anti-mouse 15 nmgold-conjugated antibody. After a final 3× wash in PBS and thenMillipore H2O, the grids were stained with 1% uranyl formate for 15seconds and imaged on a JEOL 1200 TEM.

SpyCatcher-Venus Construction and Expression

Rosetta™ cells containing pDEST14-SpyCatcher-Venus were grown up in 5 mLovernight cultures in LB at 37 C with 100 mg/L ampicillin. 500 mLcultures supplemented with ampicillin were inoculated with the overnightculture and grown up for 6 h at 37 C until an OD of 0.6.SpyCatcher-Venus expression was induced with 0.5 mM IPTG and allowed toexpress overnight at 18 C. Cells were harvested and lysed andSpyCatcher-Venus was purified on a Ni-NTA column. Protein was collected,buffer exchanged into 50 mM phosphate buffer 50 mM NaCl pH 7,concentrated and stored at −80 C until further use.

Fluorescent Biofilm Imaging

Fluorescent images were taken in epifluorescence mode on a Leica TIRFDM16000B instrument. Glass cover slips (No: 1.5) were plasma activatedfor 30 s each. Slides were immersed in 0.01 w/v % PLL solution for 2 hand then were placed in 60 C incubator for 2 h. PHL628 WT andCsgA-SpyTag(ST) cells were grown up in 20 mL cultures for 6 h at 37 C inYESCA broth containing 100 mg/L ampicillin until an OD of 0.6.Coverslips were dropped into the cultures and curli expression andbiofilm formation were induced with 0.5 mM IPTG and 3% DMSO. Cultureswere shaken at 25 C and 150 rpm for 48 h. Slides were removed from thecultures and washed 3×20 min in wash buffer (1×PBS with 0.5% Tween 20).After the washes, 0.5 mL of 1 mg/mL Venus-SpyCatcher orVenus-SpyCatcher(EQ) solution (1×PBS, 1% BSA, 0.5% Tween) was added toslides. The biofilms were incubated for 1 h and then washed 2×20 minwith wash buffer. The biofilms were then stained with SYTO 61 (10 uM)for 20 min and washed with wash buffer 2×15 min shaking at 150 rpm,Slides were then imaged in epifluorescence mode with 60× and 100× oillenses.

REFERENCES

-   1. Pasteur, L., Germ, Theory, And, Its, Applications, To, Medicine,    And, Surgery., Comptes(rendus(de(l'Academie(des(Sciences, (lxxxvi.,    1037-43, (1878).,-   2. Koch, R., Untersuchungen, Über, die, Aetiologie, der,    Wundinfectionskrankheiten, (F. C., Vogel, Leipzig, 1878).,-   3. Morrow, J. F. et al. Replication and transcription of eukaryotic    DNA in Escherichia coli. Proc Natl Acad Sci USA 71, 1743-7 (1974).-   4. Lobban, P. (Stanford University, 1972).-   5. Flemming, H C & Wingender, J. The biofilm matrix. Nat Rev    Microbiol 8, 623-33 (2010).-   6. Römling, U. & Balsalobre, C. Biofilm infections, their resilience    to therapy and innovative treatment strategies. Journal of internal    medicine (2012).-   7. Wood, T. K., Hong, S. H. & Ma, Q. Engineering biofilm formation    and dispersal. Trends in Biotechnology 29, 87-94 (2011).-   8. Singh, R., Paul, D. & Jain, R. K. Biofilms: implications in    bioremediation. Trends Microbiol 14, 38997 (2006).-   9. Perelo, L. W. Review: In situ and bioremediation of organic    pollutants in aquatic sediments. J Hazard Mater 177, 81-9 (2010).-   10. Verhagen, P., De Gelder, L. & Boon, N. Biofilm based    bioremediation strategies for the treatment of pesticide waste    streams. Commun Agric Appl Biol Sci 76, 239-43 (2011).-   11. Gross, R., Hauer, B., Otto, K & Schmid, A. Microbial biofilms:    new catalysts for maximizing productivity of long-term    biotransformations. Biotechnol Bioeng 98, 1123-34 (2007).-   12. Tsoligkas, A. N. et al. Engineering biofilms for biocatalysis.    Chembiochem 12, 1391-5 (2011).-   13. Halan, B., Buehler, K. & Schmid, A. Biofilms as living catalysts    in continuous chemical syntheses. Trends Biotechnol 30, 453-65    (2012).-   14. Chapman, M. R. et al. Role of Escherichia coli curli operons in    directing amyloid fiber formation. Science 295, 851-5 (2002).-   15. Wang, X., Smith, D. R., Jones, J. W. & Chapman, M. R. In vitro    polymerization of a functional Escherichia coli amyloid protein. J    Biol Chem 282, 3713-9 (2007).-   16. Wang, X. & Chapman, M. R. Sequence determinants of bacterial    amyloid formation. J Mol Biol 380, 570-80 (2008).-   17. Barnhart, M. M. & Chapman, M. R. Curli Biogenesis and Function.    Annual Review of Microbiology 60, 131-147 (2006).-   18. Chapman, M. R. Role of Escherichia coli Curli Operons in    Directing Amyloid Fiber Formation. Science (New York, N.Y.) 295,    851-855 (2002).-   19. Dueholm, M. S. et al. Fibrillation of the major curli subunit    CsgA under a wide range of conditions implies robust design of    aggregation. Biochemistry (2011).-   20. Hammer, N. D., Schmidt, J. C. & Chapman, M. R. The curli    nucleator protein, CsgB, contains an amyloidogenic domain that    directs CsgA polymerization. Proceedings of the National Academy of    Sciences of the United States of America 104, 12494 (2007).-   21. Nenninger, A. A. et al. CsgE is a curli secretion specificity    factor that prevents amyloid fibre aggregation. Molecular    Microbiology 81, 486-499 (2011).-   22. Nenninger, A. A., Robinson, L. S. & Hultgren, S. J. Localized    and efficient curli nucleation requires the chaperone-like amyloid    assembly protein CsgF. Proceedings of the National Academy of    Sciences of the United States of America 106, 900 (2009).-   23. Loferer, H., Hammer, M. & Normark, S. Availability of the fibre    subunit CsgA and the nucleator protein CsgB during assembly of    fibronectin-binding curliis limited by the intracellular    concentration of the novel lipoprotein CsgG. Molecular Microbiology    26, 11-23 (1997).-   24. Taylor, J. D. et al. Atomic Resolution Insights into Curli Fiber    Biogenesis. Structure 19, 1307-1316 (2011).-   25. Hanmiar, M., Arnqvist, A., Bian, Z., Olsen, A. & Normark, S.    Expression of two csg operons is required for production of    fibronectin- and congo red-binding curli polymers in Escherichia    coli K-12. Mol Microbiol 18, 661-70 (1995).-   26. Duguid, J. P., Anderson, E. S. & Campbell, L Fimbriae and    adhesive properties in Salmonellae. The Journal of pathology and    bacteriology 92, 107-138 (1966).-   27. Collinson, S. K., Parker, J., Hodges, R. S. & Kay, W. W.    Structural predictions of AgfA, the insoluble fimbrial subunit    of&lt; i&gt; Salmonella&lt;/i&gt; thin aggregative fimbriae. Journal    of Molecular Biology 290, 741-756 (1999).-   28. Dueholm, M. S. et al. Functional amyloid in Pseudomonas.    Molecular Microbiology, no-no (2010).-   29. White, A. P. et al. High efficiency gene replacement in    Salmonella enteritidis: chimeric fimbrins containing a T-cell    epitope from Leishmania major. Vaccine 17, 2150-2161 (1999).-   30. Flemming, H.-C. & Wingender, J. The biofilm matrix. Nature    Reviews Microbiology (2010).-   31. Vu, B., Chen, M., Crawford, R. J. & Ivanova, E. P. Bacterial    Extracellular Polysaccharides Involved in Biofilm Formation.    Molecules 14, 2535-2554 (2009).-   32. Freitas, F., Alves, V. D. & Reis, M. A. M. Advances in bacterial    exopolysaccharides: from production to biotechnological    applications. Trends in Biotechnology 29, 388-398 (2011).-   33. Giltner, C. L. et al. The Pseudomonas aeruginosa type IV pilin    receptor binding domain functions as an adhesin for both biotic and    abiotic surfaces. Molecular Microbiology 59, 1083-1096 (2006).-   34. Wang, X., Thou, Y., Ren, J J., Hammer, N. D. & Chapman, M. R.    Gatekeeper residues in the major curlin subunit modulate bacterial    amyloid fiber biogenesis. Proceedings of the National Academy of    Sciences 107, 163-168 (2010).-   35. Arakaki, A. A Novel Protein Tightly Bound to Bacterial Magnetic    Particles in Magnetospirillum magneticum Strain AMB-1. Journal of    Biological Chemistry 278, 8745-8750 (2002).-   36. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a    protein, through engineering a bacterial adhesin. Proceedings of the    National Academy of Sciences 109, E690-7 (2012).-   37. Thou, W., Schwartz, D. T. & Baneyx, F.o. Single-Pot    Biofabrication of Zinc Sulfide Immuno-Quantum Dots. Journal of the    American Chemical Society 132, 4731-4738 (2010).-   38. Slocik, J. M., Stone, M. O. & Naik, R. R. Synthesis of Gold    Nanoparticles Using Multifunctional Peptides. Small 1, 1048-1052    (2005).-   39. Kim, S. N. et al. Preferential Binding of Peptides to Graphene    Edges and Planes. Journal of the American Chemical Society 133,    14480-14483 (2011).-   40. Shewmaker, F. et al. The functional curli amyloid is not based    on in-register parallel beta-sheet structure. J Biol Chem 284,    25065-76 (2009).-   41. Westall, F. et al. Early Archean fossil bacteria and biofilms in    hydrothermally-influenced sediments from the Barberton greenstone    belt, South Africa. Precambrian Research 106, 93-116 (2001).-   42. Zhang, J., Zhang, E., Scott, K. & Burgess, J. G. Enhanced    electricity production by use of reconstituted artificial consortia    of estuarine bacteria grown as biofilms. Environ Sci Technol 46,    2984-92 (2012).-   43. Cegelski, L. et al. Small-molecule inhibitors target Escherichia    coli amyloid biogenesis and biofilm formation. Nat Chem Biol 5,    913-9 (2009).-   44. Lee, T. S. et al. BglBrick vectors and datasheets: A synthetic    biology platform for gene expression. J Biol Eng 5, 12 (2011).-   45. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to    several hundred kilobases. Nat Methods 6, 343-5 (2009).

TABLE 1 Length Peptide Sequence (aa) Type Function Reference HIS HHHHHH 6 Affinity Affinity Tag Bio/Technology Tag 1688, 6(11): 1321. GBPEPLQLKM  7 Substrate Graphene edge JACS 2011, 133: Binding binding14480. FLAG DYKDDDDK  8 Affinity Affinity tag Nature Biotech. Tag1988, 6: 1204. CBP HSSYVVYAFN 12 Substrate Carbon nanotubeNano. Lett. 2006, NKT Binding binding 6: 40. A3 AYSSGAPPMP 12 SubstrateGold surface Small 2005 1(11): PF Binding binding 1048. Z8 LRRSSEAHNSI12 NP ZnS quantum dot J. Mater. Chem. V templating templating2003, 13: 2414. E14 PWIPTPRPTFT 12 NP CdS quantum dot J. Mater. Chem. Gtemplating templating 2003, 13: 2414. QBP1 PPPWLPYMPP 12 SubstrateQuartz/Glass Bioinformatics WS Binding binding 2007, 23: 2816. CLP12NPYHPTIPAQS 12 Mineral Hydroxyapatite Langmuir 2011, VH templatingtemplating 27: 7620. SpyTag AHIVMVDAY 13 Protein Covalent PNAS 2012,KPTK Display capture/display 109(12): E690. of proteins CT43 CGPAGDSSGV18 NP ZnS quantum dot JACS 2010, 132: DSRSVGPC templating templating4731. MBD KCTSDQDEQF 23 Substrate Binding to Mol. Microbiol. IPKGCSKGSGBinding stainless 2006, 59(4): 1083. GSG steel surfaces AFP8 DTASDAAAA37 Substrate Ice crystal JBC 1998, AALTAANAK Binding binding273(19): 11714. AAAELTAAN AAAAAATAR Mms6 GGTIVVTGKGL 59 NP Magnetite NPJBC 2003, GLGLGLGLGA templating templating 278(10): 8745. WGPIILGVVGAGAVYAYMK SRDIESAQSDE EVELRDALA

Example 3: Programmable Biofilm-Based Materials from Engineered CurliNanofibers

Because of their role in bacterial pathogenicity and persistence, thevast majority of research on biofilms has focused on preventing theirformation and promoting their dispersal. However, this has resulted inan overlooked opportunity to develop biofilms as functional materials.Demonstrated herein is a new technology platform, Biofilm-IntegratedNanofiber Display (BIND), in which the display of functional peptides isgenetically engineered on the curli system of E. coli, the majorproteinaceous component of the biofilm matrix consisting of thinamyloidogenic nanofibers. It is contemplated herein that bacteria insuch a system can serve as a living foundry for the production,assembly, and post-processing of customized advanced biomaterials andprogrammable surface coatings. Identified herein is a fusion site in thestructural curli gene csgA that allows for the display of peptides whileretaining the ability of secreted CsgA chimeras to self-assemble intocurli fiber networks. A library of functional peptides with a range ofsizes and secondary structures were recombinantly engineered into thecurli biofilms, demonstrating the modularity and flexibility of thecurli display system. Furthermore, the kinetics and stability of theBIND nanofibers are established and it is demonstrated that the peptidesare fully displayed, as designed. Finally, the retention of peptidefunctionality in BIND biofilms was demonstrated in three broadapplications: engineered adhesion, peptide catalysis, and proteinimmobilization.

When Louis Pasteur and Robert Koch developed the germ theory of diseasein the 19th century, microbes assumed a singular role as a dominantthreat to human health1, 2. However, with advances in microbial researchand the advent of recombinant DNA technology by Lobban, Cohen, andBoyer, we entered an era where microorganisms could be geneticallymanipulated to generate biomolecules for a myriad of technologies,essentially domesticating microbial biochemistry3, 4. As describedherein, bacterial biofilms are embarking on a similar trajectory. Innature, most bacteria exist as biofilm communities, residing in aself-generated protective nanoscale scaffold of proteins, sugars,lipids, and extracellular DNA that defends against environmentalrigors5. Biofilm formation is essential for bacterial adhesion andcolonization of both natural and man-made surfaces. Characterization ofbiofilms in the mid-20th century revealed their key role in microbialpersistence and pathogenicity, which has recently led to an abundance ofresearch on biofilm prevention and dispersal.6, 7 However, these highlyevolved extracellular matrices hold untapped potential as a beneficialnanobiotechnology engineering platform. There is a significant body ofwork that investigates the use of biofilms for beneficial purposes suchas wastewater treatment8-10 and biotransformations11-13, but theseefforts focus on the use of naturally occurring organisms that happen tohave evolved various desired qualities. Efforts to rationally engineerthe structure of biofilms at the molecular level have, to our knowledge,been completely absent. To date, there exists no robust and broadtechnology for the facile engineering of biofilm components.

The approach described herein to controlling the molecular compositionof biofilm-based materials relies on the curli system—a proteinaceouscomponent of some E. coli biofilms. The curli system is composed of asmall 13-kDa-protein monomer, CsgA, that is secreted by the cell andself-assemble into highly robust amyloid nanofibers that are anchored tothe cell surface by a homologous outer-membrane protein, CsgB14-16. Theresulting curli nanofibers have a diameter of ˜7 nm and form a tangledcurly mass that encapsulates the cells.17 Curli biosynthesis is promotedby an operon that contains seven genes (csgA-G).18 Of these, CsgA is themain structural component,19 while the other proteins are involved inthe nucleation of amyloid fibers (CsgB),20 or the processing (CsgE,F),21, 22 secretion (CsgC, G)23, 24, and control of transcription(CsgD)17 of CsgA. The curli system was utilized because it exhibitsseveral features that make it amenable to the type of materialsengineering platform that are contemplated herein. First, the amyloidfibers formed by CsgA are extremely robust, being able to withstandboiling in SDS25, increasing their potential utility in harshenvironments. Second, since the extracellular fiber network is composedprimarily of a single protein, its structural features can be easilycontrolled by manipulating a single gene. Finally, although analogousextracellular amyloid systems exist in other organisms, notablySalmonella26, 27 and Pseudomonas,28 the curli system is by far the beststudied, and the fact that it occurs natively in E. coli and consists ofa single structural protein makes it highly genetically tractable.Although some of these other fimbriae systems have been investigated ascell-surface display technologies for potential vaccine deliveryagents,29 there has been no research into their use in otherbiofilm-based biotechnological applications.

Described herein is a strategy called “biofilm-integrated nanofiberdisplay” (BIND), which permits the programming of an E. coli biofilm'sfunctional properties by genetically appending functional peptidedomains to the CsgA protein. After the new CsgA-peptide is secreted andassembled, the amyloid nanofiber network displays the peptide in veryhigh density on its surface. The biofilm's function is then augmentedaccording to the sequence of the displayed peptides. It is demonstratedherein that functional peptide domains of various lengths and secondarystructures can be appended to CsgA without precluding the formation ofcurli fibers. Furthermore, the effect of peptide domain fusion on theself-assembly kinetics of the CsgA mutants is quantified. Lastly, it isdemonstrated herein that the peptide domains maintain their function inthe context of the biofilm after secretion and assembly.

Results

Design of BIND for Programmable Functionalized Biofilms.

For the design of our BIND platform, a number of considerations weretaken into account. The system has to be genetically tractable andmodular, allowing for the facile integration of any functional peptidedomain into the biofilm. This precludes the use of the polysaccharidebiopolymers that form the bulk of biofilm mass,30, 31 as their synthesisrelies on multi-enzymatic pathways that are difficult to engineer.32Proteinaceous components of biofilms known as fimbriae, which formcell-anchored nanoscale protein fibers, were selected as the scaffold ofchoice. Of the fimbriae systems in bacteria, the curli system was chosenas these amyloid nanofibers are primarily composed of a singleself-assembling protein, CsgA. This maximizes the representation of thefunctional domain in the assembled network and greatly simplifies thecomplexity of the system. Furthermore, this well-studied system isnative to E. coli, providing a wealth of genetic tools and expressiontechnologies to work with. Therefore, we first decided to test fusionsof the functional domain to the N- or C-terminus of CsgA.

C-Terminal Peptide Fusions to CsgA are Able to Form Curli Nanofibers.

Described herein is the creation of genetic fusions to the CsgA proteinthat maintain its ability to form curli fibers. This was accomplished bycreating a panel of mutants (schematically shown in FIG. 3) consistingof CsgA fused at the N- or C-terminus to a metal binding domain (MBD), apeptide domain from the Pseudomonas spp. known to bind strongly tostainless steel surfaces.33 Three variants were prepared for eachterminus: MBD is linked to CsgA either directly, or with a short (GS) orlong (GSGGSG) flanking linker. The csgA variants were cloned intoplasmids and transformed into a strain of E. coli (LSR10) missing thewild-type csgA gene, but containing the remaining curli processingmachinery.34 Therefore, upon induction, amyloid formation could beattributed solely to the heterologously engineered CsgA fusion mutants.As a readout of curli fiber production, Congo Red (CR) staining ofbacterial colonies on low-salt media, which is a standard colorimetricindicator for amyloid fibril formation, was used.14 The results of theinsertion panel show that only the C3 fusion, which has the longestlinker between the CsgA C-terminus and the MBD, is able to form anappreciable amount of amyloid fibers (FIG. 1B). Other fusions are nottolerated, as evidenced by the lack of CR staining (FIG. 1B). TEM imagesvisualizing the curli fibers that result from the CsgA fusion proteinssupport this data, with the C3 insertion site producing visiblenanofibers (FIG. 7A; wild-type curli shown for comparison in FIG. 7B).These results clearly demonstrate that it is possible to make C-terminalgenetic fusions to CsgA without inhibiting its secretion from the celland extracellular assembly into amyloid fibers.

The C3 Design Allows for Modular Incorporation of Various Peptides intoCurli Biofilms.

To test the modularity of the functional domains that can be fused tothe C-terminus of CsgA, we created a library of peptide domain fusionsranging in size from 7 to 59 amino acids while maintaining the 6 aminoacid flexible linker from C3 (Table 1). The library also representsdifferent secondary structures, as most of the peptides are not designedto exhibit any defined conformation, while MBD and Mms635 containintramolecular disulfide bonds, which should lock them into a more rigidconformation. Finally, the library members were designed to span a rangeof functions that might be potentially useful in future applications ofthe BIND system to various technologies, including capturing proteins36and binding to inorganic nanoparticles37-39 and surfaces.33 The librarymembers were cloned into LSR10 cells and probed for the formation ofcurli-based amyloid networks by CR staining. Positive CR staining (FIG.2A) and quantitative analyses (FIG. 4) for most of the library memberssuggests that small peptide fusions were tolerated by the curli exportmachinery and successfully assembled into extracellular amyloidnetworks. The only mutant for which there was no positive staining wasthe 59-amino acid Mms6 domain. This was not entirely unexpected, sinceCsgA is thought to be transported across the outer membrane by the CsgGcomplex as an unfolded conformer15. Given that the pore size of the CsgGcomplex is estimated to be ˜2 nm,24 this suggests that larger foldeddomains may not be compatible with the curli export machinery.

TEM imaging of the modified curli biofilms indicates that theCsgA-peptide fusions assemble into nano-scale fibers similar to thoseobserved for wt-CsgA (data not shown). The fibers display acharacteristic tangled curly morphology and appear to be closelyassociated with the cell surface. The TEM images were intentionallyobtained with diluted samples so that the nanostructure of the fiberscould be easily discerned. The fibers in these images that appear to befully extended are likely an artifact of the drying process duringsample preparation and do not represent the native fiber morphology.Furthermore, SEM imaging shows that the modified curli biofilms can bevery dense and several cell layers thick (data not shown) whilemaintaining a highly interconnected network of fibers between cells.

In Vitro Self-Assembly Kinetics of CsgA-Peptide Fusions.

In order to determine the effect of peptide domain fusion on theself-assembly of CsgA, several variants were selected for purificationand assembly studies in vitro. For purification purposes, theCsgA-peptide fusions were appended to a His-tag followed by anenterokinase cleavage sequence. The purification sequences were insertedin place of the Sec tag such that the proteins would not be exported tothe periplasm and after affinity purification from cell lysates, theenterokinase cleavage yielded proteins that were identical to thosesecreted by the corresponding LSR10 transformants after processing ofthe native Sec tag40. The purified proteins were monitored for theirassembly kinetics using an established thioflavin T (ThT) assay.

Functionality of Peptides Displayed with BIND. The BIND system describedherein can introduce a variety of novel functions to curli-basedbiofilms. Therefore, in addition to confirming secretion and assembly ofCsgA-peptide chimeras, it was also sought to demonstrate that the fusedpeptide domains maintain their cognate functions in the context of thefully formed biofilms. Accordingly, two peptides from Table 1 wereselected (MBD and SpyTag) and their ability to augment biofilmperformance tested. MBD was chosen because its affinity for steel shouldenhance the adhesion of curli-based biofilms to stainless steelsurfaces. To test this hypothesis, LSR10 cells expressing the CsgA-MBDmutant were grown in culture and, after induction, spotted ontostainless steel 304L coupons and allowed to dry in air. The sameprocedure was followed with cells expressing wt-CsgA and no CsgA asnegative controls. Each coupon included an array of three spots—one fromeach culture. The coupons were then subjected to vigorous washing bysubmerging them in aqueous buffer and vortexing (FIG. 5A). Biofilmscomposed of the CsgA-MBD fusion clearly withstood the washing procedure,while those expressing wt-CsgA or no CsgA were easily washed off thesurface (FIGS. 5B-5D).

These data indicate that the adhesion of curli-based biofilms tonon-natural surfaces can be artificially enhanced by appending peptidedomains that have been pre-selected to exhibit a desired function.

As a second demonstration of the utility of the BIND system, theCsgA-SpyTag mutant was investigated as a means to immobilize full-lengthproteins to the curli matrix. The SpyTag-SpyCatcher system is a recentlydeveloped strategy for protein capture that uses a CnaB2 protein thathas been split into a 13 amino acid peptide (SpyTag) and a 15-kDaprotein (SpyCatcher).36 When brought together, the two fragmentscatalyze the formation of an intermolecular isopeptide bond. Thisstrategy was used to circumvent the apparent size limitations of thecurli export machinery by enabling covalent bond formation between thecurli network and larger proteins using completely genetically encodablecomponents. Accordingly, biofilms displaying containing the CsgA-SpyTagchimera were formed on a surface-modified glass substrate using PHL628cells, an E. coli strain that has been engineered to over-produce CsgA.A SpyCatcher-Venus fusion protein was used to probe for the presence andfunctionality of the SpyTag domain. Venus is a green fluorescent proteinvariant with enhanced optical properties. The glass-immobilized biofilmswere treated with a nucleic acid stain (SYTO 61, Invitrogen) to test forthe presence of cells. Subsequently, they were treated with a solutioncontaining either SpyCather-Venus, or SpyCatcherEQ-Venus, a mutant inwhich the covalent bond formation has been abolished. Followingextensive washing steps, the biofilms were imaged using fluorescencemicroscopy. Only the proper combination of biofilms expressing theCsgA-SpyTag and treatment with SpyCatcher-Venus resulted in significantco-localization of the two fluorescent signals (FIG. 6F). Biofilmsexpressing wt-CsgA were unable to capture the fluorescent protein.Similarly, biofilms expressing CsgA-SpyTag did not exhibit greenfluorescence when treated with the non-functional SpyCatcherEQ-Venusmutant. Together these results indicate that SpyTag peptide can be fusedto CsgA and maintain its functionality after formation of the curlinetwork.

Discussion

The straightforward self-assembling system presented herein allows forthe precise molecular control of bacterial extracellular matrixcomposition by genetic engineering and establishes a platform for thecreation of functional bionanomaterials from living systems and perhapseven living functional materials. The advantages of such a syntheticbiology platform are numerous: curli fibers can be engineered to displaya variety of peptides with useful features, such as binding to orbiotemplating the synthesis of inorganic materials, enhancing biofilmadhesion to particular surfaces, or providing a scaffold-like surfacecoating for the immobilization of other biomolecules. The biofilm itselfis a “green” (i.e., environmentally friendly) material in that it ismade biosynthetically and requires no petroleum-derived raw buildingblocks. Additionally, the biofilm has the capacity to be aself-generating and self-repairing renewable material.

Surface modification and functionalization is ubiquitous in nearly allaspects of our society. However, the use of biologically-derived surfacecoatings are lacking. Life has evolved a highly efficient coatingstrategy that was an early evolutionary adaptation enabling thebacterial colonization of surfaces41. Current applications of biofilmsfor applied technology utilize naturally occurring biofilms orco-cultures of biofilm-forming bacteria42 to generate the desiredfunctionality for thin-film biocatalysis or bioremediation. Theseapplications remain limited and the adoption of biofilm-based technologyin other industries is greatly hindered by an inability to programbiofilm functionality and control the temporal dynamics of biofilmformation.

The curli system of E. coli plays a central role in host-cell adhesionof enteropathogenic strains and is critical for the formation ofbiofilms25. Curli has been extensively studied as a model system forfunctional bacterial amyloids18 as well as for the development ofbiofilm-inhibiting compounds43. It is demonstrated herein that the curlisystem can be a programmable biofilm platform as it is predominantlycomposed of a single genetically programmable unit, the self-assemblingCsgA protein.

Materials and Methods

Cell Strains and Plasmids.

All cloning and protein expression was performed in Mach1™ (Invitrogen)and Rosetta™ cells (EMD), respectively. The csgA gene was isolated fromE. coli K-12 genomic DNA and cloned into pBbE1a, a ColE1 plasmid undercontrol of the Trc promoter44. Expression vectors were constructed usingpET30a plasmids (EMD), with the native N22 region of the CsgA proteincloned immediately after the enterokinase cleavage site. Peptide insertregions were either fully synthesized (Integrated Dna Technologies) orPCR-generated by overlap extension. All cloning was performed by usingisothermal Gibson Assembly as described45 and verified by DNAsequencing.

Curli Biofilm Formation.

To produce curli, LSR10 cells or PHL628 cells were transformed withpBbE1a plasmids encoding for CsgA or CsgA-peptide fusions. As a negativecontrol, cells were transformed with empty pBbE1a plasmid. The cellswere then streaked or spotted onto YESCA-CR plates, containing 10 g/L ofcasamino acids, 1 g/L of yeast extract, and 20 g/L of agar. All mediacomponents were from Fisher. The plates were supplemented with 100 mg/mLof ampicillin, 0.5 mM of IPTG, 25 mg/mL of Congo Red and 5 mg/mL ofBrilliant Blue G250. The plates were then incubated for 48 hours at 25°C. and then imaged to determine the extent of Congo Red binding. For thespotted plates, the transformants were grown in YESCA liquid mediasupplemented with 100 mg/mL of ampicillin and 0.2 mM of IPTG for 48hours at 25° C. before spotting 20 mL onto YESCA-CR plates. This sameYESCA liquid induction procedure was used to prepare samples for CsgApurification, and electron microscopy.

Quantitative Congo Red Binding Assays.

Determination of Congo Red binding was adapted from previously publishedmethods. Briefly, transformant cultures grown on YESCA plates for 48hours at 25° C. were scraped and resuspended gently in PBS. The cellresuspention was adjusted to an OD600 of 3. To 1 mL of this, a Congo Redsolution was added to a final concentration of 0.001% and allowed toincubate at 4° C. for 1 hour. The cells were then pelleted and the 490nm absorbance of 200 μL of the supernatant was measured in a BioTek H1microplate reader. The amount of Congo Red binding was determined as thesubtractive amount of this measurement against a PBS+Congo Red control.All samples were performed in triplicate.

Chimeric CsgA Purification.

Rosetta™ cell transformants were grown in LB until mid-log phase andinduced with 0.2 mM IPTG for 3 hours. The cells were pelleted and thenfrozen at −20° C. for subsequent purification. The pellets were thawedand lysed in BugBuster Protein Extraction Reagent™ (EMD), 1 mg/mLLysozyme, 50 μg/mL DNase, and protease inhibitors (Roche). After 30minutes, the lysate was diluted into a solution of 8 guanidinehydrochloride, 250 mM NaCl, and 50 mM Tris at a pH of 7.5 and incubatedfor 16 hours to dissolve aggregates. Any insoluble mass was pelleted bycentrifugation at 18,000 rpm for 30 minutes, the clarified lysate wasfiltered through a 0.22 micron filter, and then incubated with Ni-NTAresin (Qiagen) for 2 hours. The protein-bound resin was then washed with8 guanidine hydrochloride, 250 mM NaCl, 0.1% TRITON X-100, 1 mM DTT, and50 mM Tris (pH of 7.5) and eluted with the same buffer supplemented with200 mM imidiazole. The eluate was dialyzed into EK cleavage buffer (1MUrea, 20 mM methylamine, 50 mM Tris, pH 7.5) and then incubated with 3μg of enterokinase (Roche) for 24 hours. The cleaved CsgA protein wasthen lyophilized, treated with 100 μL of HFIP to dissolve any curlifibers, and stored as a dried powder.

ThT Kinetic Assay.

Immediately before the ThT assay, the cleaved, HFIP-treated protein wasresolubilized into 8M guanidine hydrochloride, 250 mM NaCl, 0.1% TritonX-100, and 50 mM Tris at a pH of 7.5. This solution was FPLC purified ona Sephadex-G75 gel-filtration column to remove dimers and oligomers. Thefraction containing the monomeric CsgA fusions were then desalted andthe concentration determined by UV absorbance. The ThT assay wasimmediately performed with 30 μM of the CsgA fusion or wild-type proteinwith 40 μM ThT; the fluorescence was measured in a SpectramaxM2 platereader at 438ex/495em.

TEM and SEM.

Curliated wildtype or BIND cell samples were either directly taken frominduced YESCA cultures or scraped from YESCA-CR plates and resuspendedin Millipore H2O. For TEM analysis, 5 mL of the sample was spotted ontoformvar-carbon grids (Electron Microscopy Sciences), washed twice withMillipore H2O, and stained for 15 seconds with 1% uranyl formate beforeanalysis on a JEOL 1200 TEM. For SEM analysis, samples were applied toNUCLEOPORE filters under vacuum, washed with Millipore H₂O and fixedwith 2% glutaraldehyde+2% paraformaldehyde overnight at 4° C. Thesamples were then washed in Millipore H2O, dehydrated with an increasingethanol step gradient, and dried using an hexamethyldisilazane stepgradient before gold sputtering and analysis on a Zeiss Supra 55VP™FE-SEM.

Immunogold TEM.

For anti-FLAG immunogold labeling of the BIND cells displaying the FLAGtag, the cells were first adhered to the TEM grid as described above.Then, the grids were washed 3× in blocking buffer (PBS+1% BSA), floatedon a drop containing a 1:1000 dilution of primary anti-FLAG murineantibody in PBS for XX minutes, washed in blocking buffer again, andthen floated on a drop of 1:1000 diluted anti-mouse 15 nmgold-conjugated antibody. After a final 3× wash in PBS and thenMillipore H2O, the grids were stained with 1% uranyl formate for 15seconds and imaged on a JEOL1200™ TEM.

SpyCatcher-Venus Construction and Expression.

Rosetta™ cells containing pDEST14-SpyCatcher-Venus were grown up in 5 mLovernight cultures in LB at 37 C with 100 mg/L ampicillin. 500 mLcultures supplemented with ampicillin were inoculated with the overnightculture and grown up for 6 h at 37 C until an OD of 0.6.SpyCatcher-Venus expression was induced with 0.5 mM IPTG and allowed toexpress overnight at 18 C. Cells were harvested and lysed andSpyCatcher-Venus was purified on a Ni-NTA column. Protein was collected,buffer exchanged into 50 mM phosphate buffer 50 mM NaCl pH 7,concentrated and stored at −80 C until further use.

Fluorescent Biofilm Imaging.

Fluorescent images were taken in epifluorescence mode on a Leica TIRFDM16000B™ instrument. Glass cover slips (No: 1.5) were plasma activatedfor 30 s each. Slides were immersed in 0.01 w/v % PLL solution for 2 hand then were placed in 60 C incubator for 2 h. PHL628 WT andCsgA-SpyTag(ST) cells were grown up in 20 mL cultures for 6 h at 37 C inYESCA broth containing 100 mg/L ampicillin until an OD of 0.6.Coverslips were dropped into the cultures and curli expression andbiofilm formation were induced with 0.5 mM IPTG and 3% DMSO. Cultureswere shaken at 25 C and 150 rpm for 48 h. Slides were removed from thecultures and washed 3×20 min in wash buffer (1×PBS with 0.5% Tween 20).After the washes, 0.5 mL of 1 mg/mL Venus-SpyCatcher orVenus-SpyCatcher(EQ) solution (1×PBS, 1% BSA, 0.5% Tween) was added toslides. The biofilms were incubated for 1 h and then washed 2×20 minwith wash buffer. The biofilms were then stained with Syto 61 (10 uM)for 20 min and washed with wash buffer 2×15 min shaking at 150 rpm.Slides were then imaged in epifluorescence mode with 60× and 100× oillenses.

REFERENCES

-   1. Pasteur, L. Germ Theory And Its Applications To Medicine And    Surgery. Comptes rendus de l'Academie des Sciences, lxxxvi., 1037-43    (1878).-   2. Koch, R. Untersuchungen Über die Aetiologie der    Wundinfectionskrankheiten (F. C. W. Vogel, Leipzig, 1878).-   3. Morrow, J. F. et al. Replication and transcription of eukaryotic    DNA in Escherichia coli. Proc Natl Acad Sci USA 71, 1743-7 (1974).-   4. Lobban, P. (Stanford University, 1972).-   5. Flemming, H. C. & Wingender, J. The biofilm matrix. Nat Rev    Microbiol 8, 623-33 (2010).-   6. Römling, U. & Balsalobre, C. Biofilm infections, their resilience    to therapy and innovative treatment strategies. Journal of internal    medicine (2012).-   7. Wood, T. K., Hong, S. H. & Ma, Q. Engineering biofilm formation    and dispersal. Trends in Biotechnology 29, 87-94 (2011).-   8. Singh, R., Paul, D. & Jain, R. K. Biofilms: implications in    bioremediation. Trends Microbiol 14, 389¬97 (2006).-   9. Perelo, L. W. Review: In situ and bioremediation of organic    pollutants in aquatic sediments. J Hazard Mater 177, 81-9 (2010).-   10. Verhagen, P., De Gelder, L. & Boon, N. Biofilm based    bioremediation strategies for the treatment of pesticide waste    streams. Commun Agric Appl Biol Sci 76, 239-43 (2011).-   11. Gross, R., Hauer, B., Otto, K. & Schmid, A. Microbial biofilms:    new catalysts for maximizing productivity of long-term    biotransformations. Biotechnol Bioeng 98, 1123-34 (2007).-   12. Tsoligkas, A. N. et al. Engineering biofilms for biocatalysis.    Chembiochem 12, 1391-5 (2011).-   13. Halan, B., Buehler, K. & Schmid, A. Biofilms as living catalysts    in continuous chemical syntheses. Trends Biotechnol 30, 453-65    (2012).-   14. Chapman, M. R. et al. Role of Escherichia coli curli operons in    directing amyloid fiber formation. Science 295, 851-5 (2002).-   15. Wang, X., Smith, D. R., Jones, J. W. & Chapman, M. R. In vitro    polymerization of a functional Escherichia coli amyloid protein. J    Biol Chem 282, 3713-9 (2007).-   16. Wang, X. & Chapman, M. R. Sequence determinants of bacterial    amyloid formation. J Mol Biol 380, 570-80 (2008).-   17. Barnhart, M. M. & Chapman, M. R. Curli Biogenesis and Function.    Annual Review of Microbiology 60, 131-147 (2006).-   18. Chapman, M. R. Role of Escherichia coli Curli Operons in    Directing Amyloid Fiber Formation. Science (New York, N.Y.) 295,    851-855 (2002).-   19. Dueholm, M. S. et al. Fibrillation of the major curli subunit    CsgA under a wide range of conditions implies robust design of    aggregation. Biochemistry (2011).-   20. Hammer, N. D., Schmidt, J. C. & Chapman, M. R. The curli    nucleator protein, CsgB, contains an amyloidogenic domain that    directs CsgA polymerization. Proceedings of the National Academy of    Sciences of the United States of America 104, 12494 (2007).-   21. Nenninger, A. A. et al. CsgE is a curli secretion specificity    factor that prevents amyloid fibre aggregation. Molecular    Microbiology 81, 486-499 (2011).-   22. Nenninger, A. A., Robinson, L. S. & Hultgren, S. J. Localized    and efficient curli nucleation requires the chaperone-like amyloid    assembly protein CsgF. Proceedings of the National Academy of    Sciences of the United States of America 106, 900 (2009).-   23. Loferer, H., Hammar, M. & Normark, S. Availability of the fibre    subunit CsgA and the nucleator protein CsgB during assembly of    fibronectin-binding curliis limited by the intracellular    concentration of the novel lipoprotein CsgG. Molecular Microbiology    26, 11-23 (1997).-   24. Taylor, J. D. et al. Atomic Resolution Insights into Curli Fiber    Biogenesis. Structure 19, 1307-1316 (2011).-   25. Hammar, M., Arnqvist, A., Bian, Z., Olsen, A. & Normark, S.    Expression of two csg operons is required for production of    fibronectin- and congo red-binding curli polymers in Escherichia    coli K-12. Mol Microbiol 18, 661-70 (1995).-   26. Duguid, J. P., Anderson, E. S. & Campbell, I. Fimbriae and    adhesive properties in Salmonellae. The Journal of pathology and    bacteriology 92, 107-138 (1966).-   27. Collinson, S. K., Parker, J., Hodges, R. S. & Kay, W. W.    Structural predictions of AgfA, the insoluble fimbrial subunit    of&lt; i&gt; Salmonella&lt;/i&gt; thin aggregative fimbriae. Journal    of Molecular Biology 290, 741-756 (1999).-   28. Dueholm, M. S. et al. Functional amyloid in Pseudomonas.    Molecular Microbiology, no-no (2010).-   29. White, A. P. et al. High efficiency gene replacement in    Salmonella enteritidis: chimeric fimbrins containing a T-cell    epitope from Leishmania major. Vaccine 17, 2150-2161 (1999).-   30. Flemming, H.-C. & Wingender, J. The biofilm matrix. Nature    Reviews Microbiology (2010).-   31. Vu, B., Chen, M., Crawford, R. J. & Ivanova, E. P. Bacterial    Extracellular Polysaccharides Involved in Biofilm Formation.    Molecules 14, 2535-2554 (2009).-   32. Freitas, F., Alves, V. D. & Reis, M. A. M. Advances in bacterial    exopolysaccharides: from production to biotechnological    applications. Trends in Biotechnology 29, 388-398 (2011).-   33. Giltner, C. L. et al. The Pseudomonas aeruginosa type IV pilin    receptor binding domain functions as an adhesin for both biotic and    abiotic surfaces. Molecular Microbiology 59, 1083-1096 (2006).-   34. Wang, X., Zhou, Y., Ren, J. J., Hammer, N. D. & Chapman, M. R.    Gatekeeper residues in the major curlin subunit modulate bacterial    amyloid fiber biogenesis. Proceedings of the National Academy of    Sciences 107, 163-168 (2010).-   35. Arakaki, A. A Novel Protein Tightly Bound to Bacterial Magnetic    Particles in Magnetospirillum magneticum Strain AMB-1. Journal of    Biological Chemistry 278, 8745-8750 (2002).-   36. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a    protein, through engineering a bacterial adhesin. Proceedings of the    National Academy of Sciences 109, E690-7 (2012).-   37. Zhou, W., Schwartz, D. T. & Baneyx, F.o. Single-Pot    Biofabrication of Zinc Sulfide Immuno-Quantum Dots. Journal of the    American Chemical Society 132, 4731-4738 (2010).-   38. Slocik, J. M., Stone, M. O. & Naik, R. R. Synthesis of Gold    Nanoparticles Using Multifunctional Peptides. Small 1, 1048-1052    (2005).-   39. Kim, S. N. et al. Preferential Binding of Peptides to Graphene    Edges and Planes. Journal of the American Chemical Society 133,    14480-14483 (2011).-   40. Shewmaker, F. et al. The functional curli amyloid is not based    on in-register parallel beta-sheet structure. J Biol Chem 284,    25065-76 (2009).-   41. Westall, F. et al. Early Archean fossil bacteria and biofilms in    hydrothermally-influenced sediments from the Barberton greenstone    belt, South Africa. Precambrian Research 106, 93-116 (2001).-   42. Zhang, J., Zhang, E., Scott, K. & Burgess, J. G. Enhanced    electricity production by use of reconstituted artificial consortia    of estuarine bacteria grown as biofilms. Environ Sci Technol 46,    2984-92 (2012).-   43. Cegelski, L. et al. Small-molecule inhibitors target Escherichia    coli amyloid biogenesis and biofilm formation. Nat Chem Biol 5,    913-9 (2009).-   44. Lee, T. S. et al. BglBrick vectors and datasheets: A synthetic    biology platform for gene expression. J Biol Eng 5, 12 (2011).-   45. Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to    several hundred kilobases. Nat Methods 6, 343-5 (2009).

Example 4: Orthogonal Enzyme Immobilization onto Curli Fibers of E. coliBiofilm

Using biofilms for catalysis is desirable due to the their ability towithstand harsh conditions, their natural attachment to surfaces andtheir scalability. Whole cell catalysis using biofilms has limitations,however, due to the need for mass transport of substrates across thecell membrane. Enzyme display on the surface of bacteria has had limitedsuccess because of a need to co-express the enzyme with a trans-membraneor surface-displayed protein and the limited surface real estate ofbacteria. Using Biofilm Integrated Nanofiber Display, BIND, a platformas described herein was developed for covalently and site-specificallyattaching orthogonal enzymes to the extracellular matrix of E. colibiofilms. α-Amylase fused to an attachment domain, SpyCatcher, wasimmobilized onto E. coli biofilms displaying curli fibers with a capturedomain, SpyTag. It is demonstrated herein that biofilms protected theenzyme from harsh pH conditions compared to free enzyme. Furthermore,the biofilms protected the immobilized α-Amylase from denaturation inimmiscible organic solvents. Described herein is a new method of usingthe extracellular polymeric matrix of E. coli for creating versatile andcontrollable biocatalytic surfaces.

Biocatalysis provides an environmentally friendly alternative¹ tochemical synthesis with its ability to perform complex chemicaltransformations in a scalable manner². The main types of biocatalystsare whole-cell engineered microbes³, cell lysates or purified enzymes⁴Nature has evolved enzymes to be able to perform catalysis that createsstereoselective intermediates of large, complex molecules, a feat thatthe synthetic chemistry field is still trying to replicate. Furthermore,in whole-cell catalysis, multi-enzyme pathways allow the transformationof simple input molecules, such as glucose, into high value products⁵.

Despite the advantages of these biocatalysts, they have their respectivedisadvantages. Whole-cell catalysts have limitations in the types ofreactions they can catalyze due to solubility of the substrate inwater⁶, mass transfer limitations from hindered diffusion of thesubstrate or product across the cell membrane⁷, and the generation ofbyproducts or side-reactions⁴. Since the cells are not immobilized, theymust be separated from the reaction mixture to isolate the product.Immobilized enzymes do not face the problems with mass transport orisolation, however the cost of purifying enzymes on a large scale issignifican⁸ and the activity of the enzyme can be adversely affected bythe purification⁹ or the immobilization process. Due to their generalinstability, purified enzymes are frequently immobilized onto poroussurfaces when used for large-scale application, adding to the cost ofthe catalyst¹⁰. Biphasic systems have been developed to address theproblem of substrate insolubility in aqueous solvents, however thesesystems face issues with cell toxicity, biocatalyst inactivation overprolonged exposure to organic solvents and denaturation of the catalystfrom shearing upon vigorous mixing¹¹.

Biofilms are matrix-enclosed bacteria adhered to each other and/or tosurfaces or interfaces¹². They have many advantages over planktoniccells, like the ones traditionally used for whole-cell catalysis,because the bacteria in a biofilm secrete an extracellular polymericmatrix that provides protection against toxic chemicals¹³, metals¹⁴ andphysical stress¹⁵. At the same time, they retain the advantages of wholecell catalysts including stabilization of the required enzyme(s) in anatural, biological environment⁸, renewability and the ability to tuneactivity through engineering.

Current surface display technologies suffer from major limitationsincluding difficulty with co-displaying more than one enzyme and enzymecomplexes and limitations on the number of displayed enzymes due to thefinite surface real estate of the bacteria.

To address the current technological limitations, described herein is anew biofilm immobilization platform, Biofilm-Integrated NanofiberDisplay (BIND). BIND modifies the proteinacious component of E. colibiofilms, curli fibers, with functional peptides. In curli biosynthesis,curli monomer CsgA is secreted through the outer membrane transportedCsgG, anchored onto the transmembrane protein CsgB and subsequentlyself-assemble into amyloid fibers roughly 7 nm in diameter¹⁷. A numberof CsgA-functional peptide chimeras can be secreted through the curliexpression pathway. When this CsgA-peptide assembles into curli fibers,the peptides become functional handles that can be used to modify thefibers, to capture metals, nanoparticles or for adhesion¹⁸. BINDpermites the creation of catalytic surfaces through the transformationof the vast and inert polymer network of E. coli biofilm extracellularmatrices into an immobilization surface. Since the substrate and productof a reaction do not have to cross any membranes, this approach solvesthe problem of mass transport to the biocatalyst.

The Remaut group's expression system has limitations for proteins withmore innate structure—proteins containing disulfide bonds that createloops greater than the internal diameter of CsgG (˜2.5 nm), forexample¹⁹. The work described herein provides a more generalizablemethod for displaying proteins on curli fibers. As a result, theSpyCatcher-SpyTag system was employed. SpyCatcher catalyzes theformation of an isopeptide bond with the 14-amino acid SpyTag²⁰. Aspreviously shown, CsgA-SpyTag assembles into near-native curli fiberswith the SpyTag accessible for conjugation to SpyCatcher¹⁸.

It is demonstrated herein that the SpyCatcher-SpyTag system can be usedto immobilize a large enzyme, α-Amylase, onto the curli fibers of E.coli biofilms (FIGS. 8A-8D). This reaction is robust, with the abilityto form site-specific attachment between the two components, even in acomplex mixture. The activity of the enzyme on the biofilm wascharacterized using a filter-plate assay and it was shown that α-Amylaseactivity is retained under a range of pH and organic solvent incubationconditions, even when metabolic activity of the cells is lost.

Materials and Methods

Cell Strains and Plasmids.

All cloning and protein expression was performed in Mach1™ (Invitrogen)and Rosetta™ cells (EMD), respectively. E. coli csgA and csgA-SpyTaggenes were cloned into pBbE1a, a ColE1 plasmid under control of the Trcpromoter, as previously described. CsgA was expressed in YESCA media,containing 10 g/L of casamino acids (Fisher, BP1424), 1 g/L of yeastextract (Fisher, BP1422). α-Amylase was isolated from Bacilluslicheniformis ATCC 14580. SpyCatcher gene in pDEST14 was acquired fromAddgene (35044). Amylase was inserted at the N-terminus of SpyCatcherand the construct transferred to a pET28b vector, expressed in Rosetta™cells grown in Terrific Broth (Sigma T0918). The csgA deletion mutantwas PHL628-AcsgA (MG1655 malA-Kan ompR234 AcsgA). Cells were lysed usinga Misonix Probe Sonicator 4000™. Milipore PCF and hydrophilic PTFEfilter plates (PCFMSSLBPC10, MSRLN0410) and the Milipore MultiScreen™vacuum manifold setup was used for filter plate assays. For Amylaseactivity, 4-nitrophenyl-a-D-maltopentaoside (pNPMP, Sigma, 66068-38-0)was used as a substrate and α-Amylase from Bacillus licheniformis(Sigma, A3403) as a standard.

Curli Expression.

PHL628 cells were transformed with pBbE1a plasmids encoding for CsgA orCsgA-ST fusions prior to each experiment. The cells were then streakedonto YESCA plates, containing 10 g/L of casamino acids, 1 g/L of yeastextract, and 15 g/L of agar. The plates were supplemented with 100 g/mLof ampicillin. PHL628 cells were grown up in YESCA with Ampicillin untilan OD of 0.4-0.6 at 30° C. Curli expression was induced with 0.3 mMIPTG. Cultures were shaken for 18 h24 h at 25° C. and 150 rpm.

Quantitative Congo Red (CR) Binding Assays.

Determination of Congo Red (CR) binding was adapted from previouslypublished methods. Briefly, 1 mL of induced cultures grown in YESCA werepelleted at 5000 g for 10 min and resuspended gently in PBS. 150 uL of0.2 mM Congo Red solution in H2O was added to this and allowed toincubate at 25° C. for 10 min. The cells were then pelleted at 21 k gand the 490 nm absorbance of 200 μL of the supernatant was measured in aBIOTEK H1 microplate reader. The amount of Congo Red binding wasdetermined as the subtractive amount of this measurement against aPBS+Congo Red control. Congo Red absorbance was translated toconcentration using a standard curve.

Amylase-SpyCatcher Expression

Rosetta™ cells containing pET28b Amylase-Spycatcher were grown up in 5mL overnight cultures in LB at 30° C. with 100 mg/L kanamycin. IL ofterrific broth was supplemented with kanamycin to 100 mg/L, inoculatedwith the overnight culture and grown up for 5 h at 30° C. until an OD of0.4. AmylaseSC expression was induced with 0.5 mM IPTG and allowed toexpress overnight at 20° C. Cells were harvested and lysed in TBST andAmylaseSC was purified on a Ni-NTA column. Protein was collected, bufferexchanged into PBS and used within a day for experiments.

CsgA Conjugation Gels.

Starter cultures of PHL628 WT and ST were grown up ON in 100 ug/mL Amp.The starter cultures were used to grow up 50 mL of bacteria and curliproduction was induced at OD 0.6. Cells were allowed to express curlifor 24 h. Cells were spun down at 4700 g for 10 min and supernatantdiscarded. The pellets were redissolved in 3 mL PBS with proteaseinhibitors. Cells were sonicated using a probe sonicator at 15 W 5× (1min on, 0.5 min off) cycles. Cells were again spun down at 4700 g for 10min. 100 uL of the supernatant was spun down at 21000 g and the presenceof curli was checked using a CR assay. The rest of the supernatant wasincubated with previously isolated Amylase-SpyCatcher for 24 h. NaCl wasadded to the solution until 200 uM and solution was spun down at 21000 gfor 10 min. The supernatant was removed and the pellet was washed 1×1 mLH2O and spun down again at 21000 g for 10 min. Pellet and supernatentwere dried in separate tubes using a speed vac. Residue was redissolvedin 1.5 mL formic acid and 0.2 mL HFIP to break up the curli fibers intomonomers and the solvent evaporated off again. Residues were dissolvedin Laemli buffer containing 4M Urea, heated for 5 min and ran on gel at75V for 2 h.

In Vitro Amylase-SpyCatcher Activity Assay.

p-nitrophenyl-a-D-maltopentaoside (pNPMP) was choosen as the substrateto measure amylase activity because hydrolysis 4-nitrophenol (pNP) fromthe pentasaccharide can be monitored at 405 nm. The absorbance intensityof pNP is dependent on its protonation state, so we ran all reactions inPBS at pH 7.4 in a 96-well plate format.

Concentration of AmylaseSC and α-Amylase (Sigma) was measured using aBradford assay and diluted to 1.35 mM. 85 uL of 0.065-2 mM pNPMP inddH2O was added to 50 uL of protein in PBS. Activity was measured at 405nm ON in Biotek H1 microplate reader.

Curli Biofilm Filter Plate Assays.

CsgA and CsgA-ST expressing PHL628 cells were cultured for 18 h at 25°C. at 150 rpm as described above. Curli content was measured using thequantitative Congo Red binding assay. 50-100 uL of cells (normalized toCR absorption) were transferred onto Milipore PCF of PTFE membranefilter plates blocked with 2-4% BSA for 1.5 h. The media was filteredthrough using a vacuum manifold. Cells were washed 2×200 uL PBS. Cellswere incubated ON with 50 uL of AmylaseSC in PBS containing 1-2% BSAovernight. Liquid was removed using vacuum filtration and the biofilmswere washed quickly 3×150 uL 0.3% BSA in PBS and 3 more times over 90min shaking.

For activity assays in water-miscible solvents, 50 uL of 2× solventsolution in PBS was added with 50 uL of 2.5 mM pNPMP in H2O. Plates wereplaced on a desktop shaker at room temperature for 1.5-2 h. At the endof the experiment, the supernatant was vacuum filtered into a new96-well plate and 4-nitrophenol release measured at 405 nm.

For activity assays in water-immiscible solvents, biofilms wereincubated with 100-150 uL of solvent for 1 h. Solvent was removed andcells washed 2×150 uL 0.3% BSA in PBS. 50 uL of PBS and 50 uL of 2.5 mMpNPMP were added to the biofilm. Plates were placed on a desktop shakerat room temperature for 1.5 h. At the end of the experiment, thesupernatant was vacuum filtered into a new 96-well plate and pNP releasemeasured at 405 nm. Reference for relative activity is the pH 7 PBScondition.

Mts Assay.

Cell viability was tested using Promega CellTiter 96® AqueousNon-Redioactive Cell Proliferation Assay. Functionalized biofilms wereprepared as described above. Subsequent to exposure of biofilms to pH,miscible and immiscible organic solvents, biofilms were washed with PBS,incubated with Assay buffer for 1 h, filtered through and results readoptically at 490 nm. Reference for relative metabolic activity is the pH7 PBS condition.

SEM.

Scanning electron microscopy was performed on the Zeiss Ultra Plus FESEMat the Harvard University Center for Nanoscale Systems. For SEM imaging,the enzyme-bound biofilms were fixed in 2% gluteraldehyde 4%paraformaldehyde for 30 min, then washed twice with water. The filtermembranes were detached from the filter plate, dehydrated with anincreasing ethanol gradient, dried on a critical point dryer, thengold-sputtered and imaged on an FESEM (5 kV operating voltage).

Confocal Microscopy.

Confocal microscopy was performed on assayed cells and fixed cells fromPCF plate samples. Cells were incubated with DAPI for 20 min and washed4× over 2 h with 0.2% BSA in PBS. Membranes from PCF plates were cut outand membranes were placed between two cover slips to image with a LEICASP5 X MP Inverted Confocal Microscope with 63× glycerol lens.

Results and Discussion

Characterization of AmylaseSC Stability.

Only a few methods are currently used that allow site-specific, covalentattachment of proteins to polymers or surfaces. These generally involvepost-translational modification, or introduction of orthogonal chemicalunits through unnatural amino acids²¹. The SpyCatcher-SpyTag technologydescribed herein is unique in that it introduces a covalentpost-translational modification using completely geneticallyengineerable components with biologically compatible reactionconditions. SpyTag-SpyCatcher technology was selected over fusingenzymes directly to CsgA monomers because the CsgA secretion machinery(namely the transported CsgG) has been shown to have low tolerance forlarge, structured proteins¹⁹.

α-Amylase was used as the proof of concept demonstration for catalyticbiofilms because of its wide use, industrial applicability and thecommercial availability of a water-soluble colorimetric substrate. Whilemultiple proteins (127 domains, MBP, GFP) have been successfullyattached to SpyCatcher²⁰, being able to create a functionalenzyme-SpyCatcher fusion has not been shown before. When fusing twoproteins together, there is a concern that the activity of the enzymewill diminish due to destabilization by the fused domain or blocking ofthe active site. When designing our Amylase-SpyCatcher construct, a 13amino acid linker was included between the two proteins to try tomitigate such destabilization effects.

In order to test the stability of AmylaseSC, AmylaseSC activity wasexamined over a span of 64 days and at temperature ranges up to 80° C.As seen from the activity data gathered at 64 days, even at 37° C.,α-Amylase only looses 13% of its activity, and when attached toSpyCatcher, that activity loss is still relatively small at 30%. Lookingover a wider temperature range, AmylaseSC lost activity faster thanα-Amylase only above 60° C. (FIG. 14B). Since the normal operating rangeof most cell-based applications are below 60° C., it can be concludedthat at relevant temperatures, the two enzymes perform the same.

Kinetic studies of AmylaseSC were conducted in vitro in order todetermine the effect of attaching α-Amylase to SpyCatcher on activity.Michaelis-Menten analysis of the results showed that the Km/kcat valueswere nearly identical between wild-type α-Amylase and Amylase-SC (FIG.15A-15B), indicating that the fusion protein is able to catalyzereactions with the same efficiency as wild-type.

AmylaseSC Attachment to SpyTag Expressing Curli Fibers In Vitro.

In order for AmylaseSC to be able to attach to curli fibers, the SpyTagpeptide needs to be accessible. In a biofilm. the peptides may beblocked due to interaction with a surface, other bacteria, otherproteins or nearby SpyTag peptides and curli. Furthermore, attachment ofthe enzyme from a complex mixture is a desirable quality for animmobilization platform because large-scale purification of enzymes isboth expensive and time-consuming²².

In order to demonstrate that AmylaseSC can attach to CsgA-ST in arelevant complex mixture, while the curli fibers are assembled.AmylaseSC was incubated with crudely purified curli fibers and boundprotein visualized using a denaturing SDS-Page gel. Due to the harshsample preparation conditions, which involved using 8M urea and formicacid, all non-specificly bound proteins should be broken up and onlycovalently bound entities should run as one hand. As can be seen inlanes 1 and 2 on the gel (FIG. 9), a band at around 90 kDa (the combinedweight of CsgA-ST+AmylaseSC) appears in the CsgA-ST conjugation reactionprecipitate, but not in the CsgA wild type sample, illustrating covalentconjugation of AmylaseSC to CsgA-ST. Unconjugated AmylaseSC is found inthe soluble fractions.

AmylaseSC Immobilization onto Biofilms.

A 96-well filter plate setup was used to test the catalytic potential ofthe functional biofilms. Cells were grown in culture and allowed toexpress curli for 18 h before being transferred into 96-well filterplates. The biofilms were then functionalized with AmylaseSC over 24 hand reacted with pNPMP. After the desired reaction time, the solutionswere filtered into another 96-well plate and analyzed for the hydrolysisof pNP.

A seeding density of cells was chosen such that the biofilm would becomposed of a mono or bilayer of cells on the filters. This cell densitywas desirable because the MG1655 AcsgA ompR234 cells used in theseexperiment express both curli and cellulose in their extracellularmatrix, resulting in a large quantity of extracellular material thatblocks the filters when present in higher density. Confocal fluorescenceimages of the biofilms stained with DAPI are shown in FIGS. 10A-10B. Ascan be seen, the cells are surrounded by a thick matte of extracellularmaterial. Due to the compression of the fibers from the vacuumfiltration, it is difficult to distinguish between curli and celluloseon the images, however it is evident that the extracellular matrix formsa large, accessible surface area. Although this matrix looks solid onthe SEM images (data not shown), it is most likely porous in realitybecause the DAPI staining was done on the same samples prior to SEMimaging, indicating that small molecules can get through.

It was next sought to determine the amount of AmylaseSC that wouldsaturate the available SpyTag sites on the biofilm. Biofilms with around4×10⁷ cells were incubated with 20-1500 pmol AmylaseSC and the activityof the enzymes on the biofilms was measured. As shown in FIG. 11A, themaximum activity of the biofilm is reached above roughly 250 pmolAmylaseSC in the incubation buffer. It is important to note that thisdoes not accurately reflect the amount of AmylaseSC actually attached tothe biofilm—even at 20 pmol enzyme, most of the enzyme is observed inthe reaction supernatant (data not shown). Instead of seeing a saturatedsignal at the 20 pmol incubation concentration, it is hypothesized thatthe reason a normal binding curve is observed is due to the limiteddiffusion of the enzyme. That is, since the biofilm is located at thebottom of the 96-well plate, rather than being distributed throughoutthe well, not all of the AmylaseSC can find eligible CsgA-ST to bind towithin the incubation time. For the experiments in the rest of thepaper, biofilms were incubated with 750 pmol AmylaseSC to ensuresaturation of the sites.

The relationship between the amount of cells per well and the activityof the biofilm provides information about the relationship between cellcount and the amount of curli available for immobilizing AmylaseSC. Asbiofilms become thicker, there is a decrease in the extent to whichmolecules, such as nutrients, antibiotics, etc. can reach the bottomlayers of cells^(12,23). This provides biofilm with an importantevolutionary advantage over planktonic bacteria, however in the case ofthe present catalytic system, there is an inevitable tradeoff betweenbiofilm thickness and the ability of AmylaseSC to reach possibleconjugation sites and for the amylase substrate to reach catalyticsites. FIG. 11B shows that the activity of the biofilm linearlyincreases between 8×10⁶ CFU to 7×10⁷ CFU/well (upper limit due to filterclogging). This indicates that the curli that is added with eachincrease in cell count is accessible to the same extent as curlipreviously found in the biofilm. Using linear regression, with every 10⁷cells added to the biofilms in this range, an extra 4.6 nmol or 3.7% oftotal pNPMP is hydrolyzed in the present system. This number would behigher if the filters were constantly shaken or used in a flow system.

AmylaseSC Activity on Biofilms Under Various pH.

The change in ionization of charged residues on an enzyme can disruptthe enzyme's activity²⁴ by partially unfolding the protein and/ordestabilizing the active site. It was hypothesized that an enzymeimmobilized onto curli fibers would be less susceptible to activity lossdue to extreme pH because of a buffering effect created by ionizablegroups on the cells, curli fibers and other nearby proteins.

The activity of enzymes as a function of pH was measured between pH2-12. This range was chosen because it has been previously shown thatα-Amylase looses activity around pH 4 and 10²⁵. As shown in FIG. 12A,AmylaseSC attached to the biofilms is close to 100% active at pH 4 and10 while, under these conditions, α-amylase in solution looses 40% ofits activity at those pHs. Neither the solution phase nor theimmobilized enzyme shows activity below pH 3 or above pH 11, suggestingthat the environment around immobilized AmylaseSC is partially buffered.With less stable enzymes than α-amylase, the protective effect of thesebiofilms may be even more pronounced.

Metabolic activity of the biofilms was tested to determine if the samepH effect is observable on the internal enzyme activity as for AmylaseSCon the curli fibers. FIG. 12B shows that biofilms have metabolicactivity at all pHs except pH 2 and 12. The higher activity at pH 3-6than pH 7 (normalization pH) is most likely a result of stress on thecells from lower pH, which increases their metabolic activity as theytry to mitigate sub-optimal environmental conditions. A higher pHtolerance for the whole cells is reasonable because the enzymes insidecells would be better protected from denaturation than enzymes on theextracellular matrix.

AmylaseSC Activity on Biofilms in Organic Solvents.

For the successful use of catalytic biofilms, they may need to withstandconditions that are not normally beneficial for bacterial growth orenzyme stability. One major reason for this is that many small moleculeenzyme substrates cannot be dissolved in water. There have been severalways developed to attempt to circumvent this problem, includingimmobilization of enzymes onto porous scaffolds that are then used inorganic solvents²⁶⁻²⁸ and the use of two-phase aqueous-organicsystems^(29,30). In both of these cases, the organic soluble moleculebriefly enters the aqueous phase, where the enzyme is able to catalyzethe reaction, and then exits again into the organic phase.

It was hypothesized that like these two-phase aqueous-organic systems,when exposed to hydrophobic solvents, the biofilm would stay hydratedand provide a protective shell around the enzymes. To test this, thebiofilm was incubated with a panel of water-miscible and non-miscibleorganic solvents and the activity and viability of the biofilms tested.Since pNPMP is not soluble in solvents other than water, fornon-miscible solvents, the biofilms were incubated in the organics andthen replaced the solvent with PBS while measuring activity. As shown inFIG. 13A. the relative activity of AmylaseSC is only slightly affectedby incubation with non-miscible solvents, but completely disappears inmiscible solvents.

The partitioning coefficient, a measure of hydrophobicity, correlates inan s-curve manner with bioactivity³¹. In general, water-misciblesolvents have a log P<0, log P of 0˜2 corresponds to polar organiccompounds and log P>2 to mostly nonpolar compounds. For whole cellcatalysts, a log P of 2 is necessary for any activity. The resultsplotted against the partition coefficient show that enzyme activity ispreserved when biofilms are incubated in solvents with log P>0-100%activity is retained for solvents with log P>2 and, in contrast to wholecell catalysts, 70-90% activity is retained in solvents with log P0.6-0.8. This is a unique feature of the present system and points tothe potential of a broader use for the biofilm platform in organicsynthesis.

To investigate if there was a limit up to which AmylaseSC could toleratemiscible organic solvents, activity in 10-50% acetonitrile. dioxane, DMFand DMSO was examined (FIG. 13C). Even at 10% solvent, the biofilms lostbetween 30-60% of their activity and nearly all activity by 50%. Out ofthe solvents tested. DMSO caused the least activity loss.

To understand the utility of the BIND technology for variousbiocatalytic applications, it is important to know whether the cellsthemselves can stay alive at the same time as the catalyst. If the cellsdie in organic solvents, then they are unable to regenerate the curlinetwork and enzyme, if they are programmed to synthesize both. In thiscase, the curli functions as a high-surface area polymer similar tosynthetic polymer systems, except with a large number of attachmentdomains. If the cells are able to stay alive, then it is possible toimagine an integrated, renewable system in which both the curli andenzyme are regenerated and the expression of either of those componentscan be externally controlled. To understand which category the presentsystem falls into, the metabolic activity of the cells was examined as ameasure of cell viability (FIG. 13D). The most hydrophobic solvent,decane (log P=5.6), was the only organic solvent in which the cellsstayed alive after incubation.

Cell death upon incubation with organic solvents is potentially abeneficial feature of this system. however. One major challenge whenworking with biofilm reactors is the difficulty to control the growth ofthe bacteria. Generally, bacteria grow until they clog the reactor,causing the need for the whole system to be cleaned and the biofilmreplaced²². In the present system, organic solvent can be used to killthe bacteria without destroying the catalyst, effectively eliminatingthis problem.

Conclusions

Demonstrated herein is a novel platform for the immobilization of anorthogonal enzyme onto the extracellular matrix of an engineeredbiofilm. Using the BIND technology, biofilms displaying functionalhandles were created on the curli network of E. coli. Subsequently theSpyTag-SpyCatcher technology was used to site-specifically conjugateα-Amylase to the biofilms.

The novelty of the BIND system lies in the ability to create afunctionalizable polymer surface that is a scalable alternative toenvironmentally unfriendly synthetic polymers. Unlike most suchpolymers, CsgA-ST expressing curli fibers displays functional handlesthat can be easily modified by any enzyme expressed as a fusion proteinwith SpyCatcher. Like enzymes displayed on synthetic polymers,displaying enzymes using BIND allows biocatalysis on a living surfacewithout the mass transfer limitations in uniting the substrate and theenzyme in a whole cell catalyst.

Furthermore, all parts of the system are genetically controllable, fromthe synthesis of the curli fibers to the expression of theenzyme-SpyCatcher fusion protein, making this technology highlyadaptable to many applications. The BIND platform at its core is animmobilization strategy and this paper further demonstrates that BINDcan be used to display enzymes that are then protected from harshenvironmental conditions. Vast amount of research effort is placed, andwill be placed in the future, in optimizing enzymes for the catalysis ofsynthetic intermediates for applications such as pharmaceuticalsynthesis and breakdown. The BIND system eliminates the need toreengineer bacteria to incorporate these new enzymes in whole cell orbiofilm catalysis by allowing the researcher to simply switch out theenzyme attached to curli.

Another advantage of the BIND system is that the functional state of thecells is irrelevant to the catalysis. In whole cell biofilm catalysis,only a fraction of cells are performing the actual catalytic function³²,while the rest of the cells may be growing, inactive or dead.Furthermore, the metabolic activity of cells shifts during the biofilmmaturation process, so the activity is not constant during the lifetimeof the experiment³³. Since the enzymes are displayed on the externalpolymer network in BIND, their ability to catalyze a reaction isindependent of the stage of the cell cycle.

It is contemplated herein that the methods and compositions describedherein permit displaying multiple enzymes on curli, either by usingSpyCatcher-SpyTag immobilization only or by using a combination ofimmobilization domains. This would produce biofilms that can synthesizeor break down molecules through multiple enzymatic steps. As shownpreviously, clustering of sequential enzymatic steps on the nanoscalehas advantages in terms of the efficiency of the catalytic process³⁴.

Finally, development of enzyme display using BIND permits theintegration of the curli and enzyme synthesis machinery into the samebacterium. Integrating curli and enzyme synthesis machinery would allowthe enzyme-SpyCatcher fusion to be expressed and secreted by theimmobilized bacteria, eliminating the need for a separate proteinisolation step. This would lead to the creation of a completelyself-functionalizing catalytic surface. Potential applications of such atechnology would lie in many forms of ‘green’ biocatalysis, including inpharmaceutical synthesis, breakdown of pharmaceuticals in wastewater,removal of contaminants from groundwater or the creation of catalyticsurfaces for bioenergy.

REFERENCES

-   1. Sheldon, R. A. & Rantwijk, F. V. Biocatalysis for Sustainable    Organic Synthesis. Aust. J. Chem. 57, 281 (2004).-   2. Wohlgemuth, R. Modular and scalable biocatalytic tools for    practical safety, health and environmental improvements in the    production of speciality chemicals. Biocatal Biotransformation 25,    178-185 (2007).-   3. Murphy, C. D. The microbial cell factory. Org. Biomol. Chem.    (2011).-   4. Pollard, D. J. & Woodley, J. M. Biocatalysis for pharmaceutical    intermediates: the future is now. Trends in Biotechnology (2007).-   5. Eriksen, D. T., Lian, J. & Zhao, H. Journal of Structural    Biology. Journal of Structural Biology 185, 234-242 (2014).-   6. Leon, R., Fernandes, P., Pinheiro, H. M. & Cabral, J. Whole-cell    biocatalysis in organic media. Enzyme and Microbial Technology 23,    483-500 (1998).-   7. Chen, R. R. Permeability issues in whole-cell bioprocesses and    cellular membrane engineering. Appl Microbiol Biotechnol 74, 730-738    (2007).-   8. Halan, B., Buehler, K. & Schmid, A. Biofilms as living catalysts    incontinuous chemical syntheses. Trends in Biotechnology 30, 453-465    (2012).-   9. Mark, J. H. & Rebecca, J. M. Biofilms and their engineered    counterparts: A new generation of immobilised biocatalysts. Catal.    Sci. Technol. 2, 1544-1547 (2012).-   10. Zhou, Z. & Hartmann, M. Recent Progress in Biocatalysis with    Enzymes Immobilized on Mesoporous Hosts. Top Catal 55, 1081-1100    (2012).-   11. Wang, Z., van Oers, M. C. M., Rutjes, F. P. J. T. & van    Hest, J. C. M. Polymersome Colloidosomes for Enzyme Catalysis in a    Biphasic System. Angew. Chem. Int. Ed. 51, 10746-10750 (2012).-   12. Costerton, J. W., Lewandowski, Z., Caldwell, D. E.,    Korber, D. R. & Lappin-Scott, H. M. Microbial biofilms. Annu. Rev.    Microbiol. 49, 711-745 (1995).-   13. Fang, H. H. P., Xu, L.-C. & Chan, K.-Y. Effects of toxic metals    and chemicals on biofilm and biocorrosion. Water Res. 36, 4709-4716    (2002).-   14. Harrison, J. J., Ceri, H. & Turner, R. J. Multimetal resistance    and tolerance in microbial biofilms. Nature Publishing Group 5,    928-938 (2007).-   15. Gross, R., Lang, K., BÃ¼hler, K. & Schmid, A. Characterization    of a biofilm membrane reactor and its prospects for fine chemical    synthesis. Biotechnol. Bioeng. n/a-n/a (2009). doi:10.1002/bit.22584-   16. van Bloois, E., Winter, R. T., Kolmar, H. & Fraaije, M. W.    Decorating microbes: surface display of proteins on Escherichia    coli. Trends in Biotechnology 29, 79-86 (2011).-   17. Chapman, M. R. Role of Escherichia coli Curli Operons in    Directing Amyloid Fiber Formation. Science 295, 851-855 (2002).-   18. Chen, A. Y. et al. Synthesis and patterning of tunable    multiscale materials with engineered cells. Nat Mater (2014).    doi:10.1038/nmat3912-   19. Van Gerven, N. et al. Secretion and functional display of fusion    proteins through the curli biogenesis pathway. Molecular    Microbiology 91, 1022-1035 (2014).-   20. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a    protein, through engineering a bacterial adhesin. Proc. Natl. Acad.    Sci. U.S.A. 109, E690-7 (2012).-   21. Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry:    fishing for selectivity in a sea of functionality. Angew. Chem. Int.    Ed. Engl. 48, 6974-6998 (2009).-   22. Qureshi, N., Annous, B. A., Ezeji, T. C., Karcher, P. &    Maddox, I. S. Microbial Cell Factories|Full text|Biofilm reactors    for industrial bioconversion processes: employing potential of    enhanced reaction rates. Microb Cell Fact 4, 24 (2005).-   23. Stewart, P. S. Diffusion in Biofilms. J. Bacteriol. 185,    1485-1491 (2003).-   24. Di Russo, N. V., Estrin, D. A., Marti, M. A. & Roitberg, A. E.    pH-Dependent Conformational Changes in Proteins and Their Effect on    Experimental pKas: The Case of Nitrophorin 4. PLoS Comput Biol 8,    e1002761 (2012).-   25. Nielsen, J. E., Borchert, T. V. & Vriend, G. The determinants of    α-amylase pH-activity profiles. Protein Engineering 14, 505-512    (2001).-   26. Sinisterra, J. V. & Dalton, H. in Progress in Biotechnology 11,    416-423 (Elsevier, 1996).-   27. Hertzberg, S., Kvittingen, L., Anthonsen, T. & Skjåk-Bræk, G.    Alginate as immobilization matrix and stabilizing agent in a    two-phase liquid system: Application in lipase-catalysed reactions.    Enzyme and Microbial Technology 14, 42-47 (1992).-   28. Kawakami, K., Tsuruda, S. & Miyagi, K Immobilization of    microbial cells in a mixed matrix of silicone polymer and calcium    alginate gel: epoxidation of 1-octene by Nocardia corallina B-276 in    organic media. Biotechnol. Prog. 6, 357-361 (1990).-   29. Munoz, R., Daugulis, A. J., Hernández, M. & Quijano, G.    Biotechnology Advances. Biotechnology Advances 30, 1707-1720 (2012).-   30. Brink, L. & Tramper, J. Modelling the effects of mass transfer    on kinetics of propene epoxidation of immobilized Mycobacterium    cells: 1. Pseudo-one-substrate conditions and negligible product    inhibition. Enzyme and Microbial Technology 8, 281-288 (1986).-   31. Laane, C., Boeren, S., Vos, K. & Veeger, C. Rules for    optimization of biocatalysis in organic solvents. Biotechnol.    Bioeng. 30, 81-87 (1987).-   32. Qureshi, N., Paterson, A. H. J. & Maddox, I. S. Model for    continuous production of solvents from whey permeate in a packed bed    reactor using cells of Clostridium acetobutylicum immobilized by    adsorption onto bonechar. Appl Microbiol Biotechnol 29, 323-328    (1988).-   33. Jones, K. & Bradshaw, S. B. Biofilm formation by the    enterobacteriaceae: a comparison between Salmonella enteritidis,    Escherichia coli and a nitrogen-fixing strain of Klebsiella    pneumoniae. J. Appl. Bacteriol. 80, 458-464 (1996).-   34. Schoffelen, S. & van Hest, J. C. M. Multi-enzyme systems:    bringing enzymes together in vitro. Soft Matter 8, 1736 (2012).

Example 5: Programmable Biofilm-Based Materials from Engineered CurliNanofibers

Self-assembling living systems that are autonomously generating,renewable, and programmable are the next generation of advancedbiomaterials. Described herein is “Biofilm-Integrated Nanofiber Display”(BIND) as a strategy for the programmable functionalization of the E.coli biofilm matrix by genetically appending various peptides to theamyloidogenic protein CsgA(I), a biofilm proteinaceous component. CsgAfusion proteins are successfully secreted by the cellular exportmachinery, self-assemble into amyloid nanofiber networks, and displaythe peptide of interest in high density. The displayed peptide domainsconfer various non-natural functions to the biofilms, including adhesionto specific surfaces, nanoparticle templating, protein immobilization,or a combination thereof. BIND is a novel strategy for the broadfunctionalization of biofilms and demonstrates the utility of biofilmsas a designable biomaterial.

In the last century, advances in our understanding of bacterial systemshave expanded the role of the microbe from being regarded solely as ahealth threat to being exploited as genetically programmable factoriesfor the production of biomolecules and chemicals. Bacterial biofilms areembarking on a similar trajectory vis-à-vis functional advancedmaterials. The majority of bacteria in the natural world exist asbiofilms: organized communities of cells ensconced in a network ofextracellular polysaccharides, proteins, and other biomolecularcomponents(2). This extracellular matrix protects bacteria fromenvironmental rigors and mediates substrate adhesion, thus promotingmicrobial persistence and pathogenicity.

Hence, most biofilm research has focused their eradication due to thenegative roles biofilms play in clinical infection. Described herein isthe domestication of biofilms as a platform for a programmable andmodular self-assembling nanomaterial, with the bacterium serving as aliving foundry for the synthesis of its raw building blocks, itsassembly into higher order structures, and its maintenance over time.While there has been limited investigation into the use of naturallyoccurring biofilms for beneficial purposes such as energy generation(3),wastewater treatment(4) and biotransformations(5), their widespread useis hindered by a lack of methods to rationally engineer biofilmformation, morphology, adhesion, and function. Any engineering ofbiofilms to date has focused on altering the cellular populations ratherthan the biofilm material itself. Although methods exist to displaypeptides and proteins on various extracellular scaffolds(6), efforts torationally engineer the molecular structure and properties of thebiofilm matrix have, to our knowledge, been completely absent.

The present approach, described herein, to engineering the biofilmextracellular matrix for practical applications focuses on the curlisystem—the primary proteinaceous structural component of E. colibiofilms. Curli are highly robust functional amyloid nanofibers with adiameter of ˜7 nm that form a tangled network encapsulating the cells.Curli are formed from the extracellular self-assembly CsgA, a smallsecreted 13 kDa protein. A homologous outer-membrane protein, CsgB,nucleates CsgA assembly and also anchors the nanofibers to the bacterialsurface. The curli biosynthetic operon contains seven genes (csgA-G)(I),whose products mediate the processing (CsgE, F), secretion (CsgC, G),and transcriptional regulation (CsgD) of CsgA and CsgB.

The curli system exhibits numerous features that make it an idealplatform for the type of materials engineering by way of syntheticbiology described herein. First, since the curli nanofiber is composedprimarily of one protein, it presents a tractable entry point towardscreating a large diversity of biofilm extracellular matrices withconventional genetic engineering methods. In contrast, it would be moredifficult to engineer the exopolysaccharide component of biofilms, aspolysaccharide synthesis is often tied to multi-step pathways with alimited tolerance for chemically diverse monomers compared to theprotein synthetic machinery. Second, the functional amyloid fibersformed by CsgA are extremely robust, being able to withstand boiling indetergents(7) and extended incubation in solvents, increasing theirpotential utility in harsh environments. Similar amyloid nanofibers havebeen shown to have a strength comparable to steel and a mechanicalstiffness comparable to silk(8), suggesting that biofilms with highamyloid content may be able to withstand mechanically demandingenvironments. Third, functional amyloid fibrils are abundant in manynaturally occurring bacterial biofilms and can constitute up to 10-40%of the total biovolume(9), suggesting that curli can be artificiallyengineered to comprise a significant portion of the biofilm. Inaddition, although analogous extracellular functional amyloids areproduced by many bacteria, the curli system is the best studied and isnative to the canonical model bacteria, making it an attractive startingplatform for the development of engineered materials. Lastly, recentfindings have shown that the curli system can efficiently exportamyloidogenic polypeptides and was capable of expressing a functionalCsgA-camelid antibody fragment fusion, demonstrating that the curlisystem can be used in a broad and modular way for the display offunctional peptides throughout the E. coli biofilm matrix (10, 11).

The BIND system enables the precise genetic programming of the E. colibiofilm matrix by fusing functional peptide domains to the CsgA protein(FIG. 16A). It is demonstrated herein that the chimeric CsgA variantsare secreted by the native cellular export machinery and assemble intonetworks of curli fibers that resemble the wild-type system. It is alsodemonstrated that this technique is compatible with a wide range ofpeptide domains of various lengths and secondary structures. Lastly, itis demonstrated that the peptide domains maintain their function aftersecretion and assembly and confer artificial functions to the biofilm asa whole. In three proof-of-concept experiments, the ability to programdifferent functions into the biofilm is highlighted: specific adhesionto an abiotic surface, nanoparticle templating, and site-specificcovalent immobilization of an arbitrary functionalized recombinantprotein.

In order to determine suitable fusion points to append peptides to CsgA,a library (FIG. 3) was generated consisting of N- and C-terminal fusionsto a peptide domain known to bind strongly to stainless steelsurfaces(12). Terminal fusions were chosen to allow for the integrationof both linear and circularly constrained peptides. Three variants wereprepared for each terminus with varying flexible linker lengths. ThecsgA variants were expressed in a csgA deletion strain of E. coli(LSR10) that retains the remaining curli processing machinery undernative regulation(13). This strain does not produce flagella, cellulose,or LPS Opolysaccharides(14-16). Thus any extracellular fibers could beattributed solely to the selfassembly of heterologously engineered CsgAfusion mutants. An amyloid-staining colorimetric dye, Congo Red (CR),was used to determine the extent of curli production for the variousmutants. Based on this assay, only the C3 fusion site with the longestC-terminal linker between CsgA and MBD was able to form an appreciableamount of amyloid fibers (FIG. 1B). It is possible that the N-terminalfusions inhibited cellular export due to their proximity to theCsgGspecific export recognition sequence. Scanning (SEM, FIG. 16M) andtransmission electron microscopy (TEM, Fig. FIG. 20M) characterizationof the C3 mutant curli fibers confirmed that they exhibited morphologysimilar to the wild-type CsgA fibers.

Having identified a suitable fusion site, a library of 12 peptide domainfusions was created to test the effect of peptide length and structureon secretion and assembly. The library members ranged in length from 7to 59 amino acids, encoded a wide variety of functions(17-28), and werefused to CsgA C-terminus using the six-amino acid flexible linker (Table1, FIG. 17). The library members were cloned into LSR10 cells and probedfor the formation of curli by CR staining. Quantitative differences incurli production between library members were monitored by measuring thestaining intensity of transformants spotted on CR plates (FIG. 16B). SEM(FIG. 16C-16P) and TEM (FIGS. 20A-20N) of the modified curli biofilmssupports the CR data for the production of extracellular amyloid.Overall, most small peptide fusions were tolerated by the curli exportmachinery and could successfully assemble into amyloid networks.Immunostaining of BIND biofilms expressing the CsgA-FLAG mutant withanti-FLAG antibody confirmed the presence and accessibility of thepeptide domain (FIGS. 21A-21B). The only mutant for which there was nopositive CR staining was the 59-amino acid Mms6 protein domain,confirming previous findings that polypeptides with long sequences orinherent structure may not be exported efficiently through the CsgGouter membrane transporter, which has a pore size of 2 nm (10, 29). Forthe curli-producing strains, the CsgA-peptide fusions assemble intonanoscale fibers similar in morphology to wt-CsgA (FIG. 16C-16O). Thefibers display a characteristic tangled morphology and appear to beclosely associated with the cell surface in meshlike networks,suggesting that peptides of arbitrary sequence and function could bedisplayed on the surface of curli fibers. Some of the BIND variants,such as the FLAG-BIND, exhibited the ability to form extensive thin,fabric-like 2D meshes unseen in normal curli biofilms, suggesting thatthe platform can also be manipulated to alter the macromoleculararchitecture of biofilms (FIG. 22).

The true value of the BIND system is in its ability to perform as anactive surface coating whose function can be genetically programmed in amodular fashion. As a demonstration of some of these capabilities, threepeptides were selected from Table 1 (MBD, A3 and SpyTag) and theirability to introduce new functions to curli-producing biofilms tested,specifically, the ability to enhance adhesion to abiotic surfaces, tobiotemplate the growth of inorganic nanoparticles, and to covalentlyimmobilize full-length proteins. For these studies PHL628, a csgAdeletion strain which overproduces the curli processing machinery and isalso able to produce CR-positive biofilms was used (FIG. 4) (30).

In order to make BIND an efficient platform for developing interfacialmaterials, it will be critical to tune the nanofiber adhesion tospecific abiotic surfaces. As an example of this capability, theadhesion of E. coli cells displaying MBD to 304L stainless steel, themost versatile and widely used steel alloy, was tested. PHL628 cellsexpressing the CsgA-MBD mutant were spotted onto 304L coupons, allowedto adhere for 48 hours, and then vigorously washed in aqueous buffer(FIG. 5A) to remove non-specifically bound cells. Biofilms composed ofthe CsgA-MBD fusion withstood the washing procedure, while thoseexpressing wt-CsgA or no CsgA were easily washed off the surface (FIGS.18B-18E). This result demonstrates that BIND programming using MBD issufficient to impart adhesive function to biofilms. The modularity ofthe BIND platform lends itself to a plug-and-play approach to the designof biofilm adhesion for applications in bioremediation or chemicalsynthesis, where non-specific biofilm growth is viewed as adisadvantage. This capability will be particularly useful inapplications where patterned surfaces are used to spatially controlbiofilm formation, or where it is desired to localize biofilm growth tospecific materials, as is often the case in industrial bioreactors.

Peptide binding to surfaces can also be used to promote materialstemplating, which is demonstrated herein using BIND composed of aCsgA-A3 fusion. The A3 peptide was previously developed by phage displayto bind silver and has been shown to control the templating of silvernanoparticles. The A3-BIND biofilms demonstrate an enhanced ability tobind to growing silver nanoparticles from a solution of AgNO3 incontrast to the wildtype biofilm (FIGS. 18D-18E). In addition todisplaying short peptides, it was reasoned that the utility of the BINDsystem would be greatly expanded if it could be used to display fullproteins of arbitrary length and dimensions to program the biofilm withartificial catalytic, electron transport, or sensing capabilities. Acompletely genetically encodable strategy was employed (FIG. 19A) tocovalently immobilize proteins onto the BIND network, using asplit-adhesin system in which a 13-amino acid peptide (SpyTag) forms anisopeptide bond with a 15-kDa protein (SpyCatcher) (31). Accordingly,biofilms displaying the CsgA-SpyTag chimera were grown on a glasssubstrate using PHL628 cells and formed a characteristic curli networkwhen either wt-CsgA or CsgA-SpyTag were expressed (FIGS. 19B-19G). ASpyCatcher-Venus fusion protein was used to probe for the presence andfunctionality of the SpyTag domain. Treatment with eitherSpyCatcher-Venus or a non-functional mutant (SpyCatcherEQ-Venus)revealed that only biofilms expressing CsgA-SpyTag were able to bindSpyCatcher-Venus (FIG. 6F). These results confirm that the SpyTagpeptide can be fused to CsgA and maintain its functionality afterformation of the curli network. To further simplify the immobilizationprocess, an unpurified cell lysate containing the SpyCatcher fusionprotein was used in lieu of purified protein, and obtained similarresults (data not shown), thus demonstrating the binding specificitybetween the CsgASpyTag curli network and its cognate SpyCatcher fusionprotein, even in complex mixtures. This feature of BIND will beespecially useful in the area of biocatalysis, for the development of anefficient immobilization process for the reuse of an enzyme.

Described herein is an elegant strategy using the BIND platform, wherebythe fabrication of the nanofiber scaffold, biosynthesis of the enzyme,and immobilization reaction could all be accomplished by a singleengineered bacterial strain without any purification steps.

A key aspect of BIND is that by virtue of the random extracellularself-assembly of curli fibers, the expression of different CsgA fusionswill result in a multifunctional biofilm surface. Thus, a BIND structurecan be programmed for any combination of adhesion, display, moleculartemplating, or protein immobilization. This characteristic isdemonstrated herein by coculturing CsgA-FLAG and CsgA-SpyTag to producea bifunctional BIND biofilm that can display the FLAG tag as well asimmobilize GFP through the SpyTag-SpyCatcher system (data not shown).

Demonstrated herein is a strategy for the rational molecular design of amicrobial extracellular matrix component with the purpose of introducingnew function into a biofilm. These results demonstrate that the curlisystem in E. coli is capable of secreting and assembling a variety ofchimeric CsgA-peptide constructs that can self-assemble into anamyloid-based extracellular matrix. The fused peptide domains aredisplayed in high density on the network surface and maintain theirfunction even after assembly. It is demonstrated herein that threedistinct non-natural functions (adhesion to steel surfaces, silvernanoparticle templating, and covalent protein immobilization) can beintroduced modularly into E. coli biofilms based on the predeterminedfunctions of various engineered peptide sequences. Importantly, eachfunctional demonstration was accomplished without the need for systemre-optimization, suggesting that other sequences can easily beincorporated into the present system to access materials with a range ofnon-natural functions, and even multiple new functions at once. BINDlends itself to the rapid development of interfacial nanomaterials withfunctions that can be drawn from the diverse repertoire of knownpeptides and proteins. These biofilm-based materials can be used in awide range of environments that may or may not be conducive to cellularsurvival. In hospitable environments, the encapsulated cells of thebiofilm may be induced to self-regenerate or heal the material overtime, or alter the material in response to environmental cues. However,in harsher environments, the robust engineered matrix may function onits own without the need for maintenance. The methods and compositionsdescribed herein can be used to introduce new function to many othermicrobial biofilms with analogous functional amyloids (e.g. Salmonella,Pseudomonas, Bacillus spp.) to capitalize on the particular features ofeach wild-type strain. Given that the engineered bacteria proliferaterapidly and require no petroleum-derived raw building blocks in order tobiosynthesize the external matrix, BIND may be useful as a scalable and“green” approach to fabricating customized interfacial materials acrossa wide range of size scales and environments.

Materials and Methods

Cell Strains and Plasmids.

All cloning and protein expression was performed in Mach1™ (Invitrogen)and Rosetta™ cells (EMD), respectively. The csgA gene was isolated fromE. coli K-12 genomic DNA and cloned into pBbE1a, a ColE1 plasmid undercontrol of the Trc promoter. Peptide insert regions were either fullysynthesized (Integrated DNA Technologies) or PCR-generated by overlapextension. All cloning was performed using isothermal Gibson Assemblyand verified by DNA sequencing.

Curli Biofilm Formation.

To produce curli, LSR10 cells or PHL628 cells were transformed withpBbE1a plasmids encoding for CsgA or CsgA-peptide fusions. As a negativecontrol, cells were transformed with empty pBbE1a plasmid. The cellswere then streaked or spotted onto YESCA-CR plates, containing 10 g/L ofcasamino acids, 1 g/L of yeast extract, and 20 g/L of agar. These plateswere supplemented with 100 μg/mL of ampicillin, 0.5 mM of IPTG, 25 μg/mLof Congo Red and 5 μg/mL of Brilliant Blue G250. The plates were thenincubated for 48 hours at 25° C. and then imaged to determine the extentof Congo Red binding. For the spotted plates, the transformants weregrown in YESCA liquid media supplemented with 100 μg/mL of ampicillinand 0.2 mM of IPTG for 48 hours at 25° C. before spotting 20 μL ontoYESCA-CR plates.

TEM and SEM.

Curliated wildtype or BIND cell samples were either directly taken frominduced YESCA cultures or scraped from YESCA-CR plates and resuspendedin Millipore H2O. For TEM analysis, 5 μL of the sample was spotted ontoformvar-carbon grids (Electron Microscopy Sciences), washed withMillipore H2O, and stained with 1% uranyl formate before analysis on aJEOL 1200 TEM. For SEM analysis, samples were applied to Nucleoporefilters under vacuum, washed with Millipore H₂O and fixed with 2%glutaraldehyde+2% paraformaldehyde overnight at 4° C., followed byfixation in 1% osmium tetroxide. The samples were then washed inMillipore H2O, dehydrated with an increasing ethanol step gradient,followed by a hexamethyldisilazane step gradient before gold sputteringand analysis on a Zeiss Supra55VP™ FE-SEM.

Immunogold TEM.

For anti-FLAG immunogold labeling of the BIND cells displaying the FLAGtag, the cells were first adhered to nickel TEM grids as describedabove. Then, the grids were washed 3× in blocking buffer (PBS+1% BSA),floated face-down on a drop containing a 1:1000 dilution of primaryanti-FLAG murine antibody in PBS for 30 minutes, washed in blockingbuffer again, and then floated on a drop of 1:1000 diluted anti-mouse 15nm gold-conjugated antibody for 30 minutes. After a final 3× wash in PBSand then Millipore H2O, the grids were stained with 1% uranyl formatefor 15 seconds and imaged on a JEOL1200™ TEM.

MBD-BIND Binding to 304L Stainless Steel Coupons.

Steel alloy 304L coupons (Alabama Specialty Products, Inc.) were cleanedwith fine-grit sandpaper, acetone, Millipore water, sonicated in 1M NaOHfor 1 hour at 80° C., washed again with Millipore water, and finallyrinsed with acetone before air-drying. PHL628 csgA transformants weregrown in YESCA media as described above and induced by adding 0.5 mMIPTG and 3% DMSO for 48 hours at 25° C., 150 rpm. Cell cultures werenormalized to an OD600 of 1 and 20 μL was spotted onto a 304L coupon.The spotted coupon was placed in a sterile petri dish and placed in 4 Cto allow attachment and minimize evaporation. After 48 hours, thecoupons were rinsed briefly with PBS, placed in a tube filled with PBS,and vortexed 3× for 30 seconds at a vortex setting of 5. The couponswere then fixed and SEM imaged according to the protocols describedabove.

Silver Nanoparticle Templating.

PHL628 csgA cells were transformed with wild-type CsgA or CsgA-A3expressing plasmids and induced with 0.2 mM IPTG in YESCA brothcontaining 100 μg/mL carbenicillin for 48 hours. The cells and curliwere isolated by pelleting and then resuspended in PBS+CM.Nickleformvar/carbon TEM grids were floated on drops of theseresuspended samples, washed twice with PBS+CM, thrice with mpH2O, andthen incubated on a drop containing 147 mM AgNO3 for 4 hours. The gridswere then washed thrice with mpH2O and negatively stained and analyzedby TEM as described above.

Biofilm Fluorescence Microscopy Imaging.

PHL628 csgA cells transformed with control, wild-type CsgA, andCsgA-SpyTag expressing plasmids were grown up in 20 mL YESCA brothcontaining 100 μg/mL ampicillin at 30° C. until an OD of 0.6.Plasma-activated and PLL-functionalized coverslips were placed into thecultures and curli expression and biofilm formation were induced byadding 0.5 mM IPTG and 3% DMSO. Cultures were grown at 25° C. and 150rpm for 48 hours. Slides were removed from the cultures and washed 3×20min in wash buffer (1×PBS+0.5% Tween 20), shaking at 150 rpm. After thewashes, 0.5 mL of 1 mg/mL Venus-SpyCatcher or Venus-SpyCatcher(E77Q)solution (in PBS+1% BSA+0.5% Tween) was added to slides. The biofilmswere incubated for 1 hour and then washed 2×20 min with wash buffer. Thesamples were then stained with SYTO-61 (10 μM) for 20 min and washedwith wash buffer 2×15 min shaking at 150 rpm. Slides were then imaged inepifluorescence mode on a Leica Tirf DM16000B™ at 60× and 100×. For themultifunctional BIND experiments, cells at an initial OD600 of 2.5 werecultured in MatTek glass-bottom dishes for 72 hours under inducingconditions (YESCA/0.5 mM IPTG/100 ng/mL carbenicillin/3% DMSO). Thebiofilms were then washed 3×10 min in PBST, blocked with 1% BSA in PBSTfor 1 hour, and incubated with Venus-SpyCatcher containing clarifiedcell lysate for 1 hour. The dishes were then extensively washed with0.1% BSA+PBST under gentle shaking before incubation with an anti-FLAGDyLight® 680 antibody (Pierce) for 1 hour. The samples were washed asbefore with 0.1% BSA+PBST, fixed with 2% glutaraldehyde+2%paraformaldehyde in 0.1M sodium cacodylate buffer for 15 minutes, andthen incubated in PBS+10 mM glycine overnight at 4° C. to eliminateautofluorescence. All multifunctional BIND samples were analyzed onLeica SP5 X MP™ Inverted Confocal Microscope.

SpyCatcher-Venus Construction and Expression.

Rosetta™ cell transformants containing pDEST14-SpyCatcher-Venus wereused to inoculate 500 mL cultures supplemented with 100 g/mL ampicillinand grown for 6 hours at 37° C. until an OD of 0.6. SpyCatcher-Venusexpression was induced with 0.5 mM IPTG and allowed to express overnightat 18° C. Cells were harvested and lysed and SpyCatcher-Venus waspurified on a Ni-NTA column. Protein was collected, buffer exchangedinto 50 mM phosphate buffer/50 mM NaCl, pH 7, concentrated and stored at−80° C. until further use. The E77Q mutant was purified in a similarmanner.

REFERENCES

-   1. M. R. Chapman, L. S. Robinson, J. S. Pinkner, R. Roth, J.    Heuser, M. Hammar, S. Normark, S. J. Hultgren, Role of Escherichia    coli curli operons in directing amyloid fiber formation. Science    295, 851-855 (2002); published online Epub February (295/5556/851    [pii] 10.1126/science.1067484).-   2. H.-C. Flemming, J. Wingender, The biofilm matrix. Nature Reviews    Microbiology, (2010); published online Epub August 02    (10.1038/nrmicro2415).-   3. B. E. Logan, Exoelectrogenic bacteria that power microbial fuel    cells. Nature Reviews Microbiology 7, 375-381 (2009).-   4. R. Singh, D. Paul, R. K. Jain, Biofilms: implications in    bioremediation. Trends Microbiol 14, 389-397 (2006); published    online Epub September (10.1016/j.tim.2006.07.001).-   5. B. Halan, K. Buehler, A. Schmid, Biofilms as living catalysts in    continuous chemical syntheses. Trends Biotechnol 30, 453-465 (2012);    published online Epub September (10.1016/j.tibtech.2012.05.003).-   6. C. G. Ullman, L. Frigotto, R. N. Cooley, In vitro methods for    peptide display and their applications. Briefings in Functional    Genomics 10, 125-134 (2011); published online Epub June 30    (10.1093/bfgp/e1r010).-   7. M. Hammar, A. Arnqvist, Z. Bian, A. Olsen, S. Normark, Expression    of two csg operons is required for production of fibronectin- and    congo red-binding curli polymers in Escherichia coli K-12. Mol    Microbiol 18, 661-670 (1995); published online Epub November (-   8. J. F. Smith, T. P. Knowles, C. M. Dobson, C. E. Macphee, M. E.    Welland, Characterization of the nanoscale properties of individual    amyloid fibrils. Proc Natl Acad Sci USA 103, 15806-15811 (2006);    published online Epub October (10.1073/pnas.0604035103).-   9. P. Larsen, J. L. Nielsen, D. Otzen, P. H. Nielsen, Amyloid-like    adhesins produced by floc-forming and filamentous bacteria in    activated sludge. Appl Environ Microbiol 74, 1517-1526 (2008);    published online Epub March (10.1128/AEM.02274-07).-   10. N. Van Gerven, P. Goyal, G. Vandenbussche, M. De Kerpel, W.    Jonckheere, H. De Greve, H. Remaut, Secretion and functional display    of fusion proteins through the curli biogenesis pathway. Mol    Microbiol, (2014); published online Epub January    (10.1111/mmi.12515).-   11. V. Sivanathan, A. Hochschild, Generating extracellular amyloid    aggregates using E. coli cells. Genes Dev 26, 2659-2667 (2012);    published online Epub December (10.1101/gad.205310.112).-   12. C. L. Giltner, E. J. van Schaik, G. F. Audette, D. Kao, R. S.    Hodges, D. J. Hassett, R. T. Irvin, The Pseudomonas aeruginosa type    IV pilin receptor binding domain functions as an adhesin for both    biotic and abiotic surfaces. Molecular Microbiology 59, 1083-1096    (2006); published online Epub March    (10.1111/j.1365-2958.2005.05002.x).-   13. X. Wang, Y. Zhou, J. J. Ren, N. D. Hammer, M. R. Chapman,    Gatekeeper residues in the major curlin subunit modulate bacterial    amyloid fiber biogenesis. Proceedings of the National Academy of    Sciences 107, 163-168 (2010); published online Epub February 05    (10.1073/pnas.0908714107).-   14. M. J. Casadaban, Transposition and fusion of the lac genes to    selected promoters in Escherichia coli using bacteriophage lambda    and Mu. J Mol Biol 104, 541-555 (1976); published online Epub July (-   15. D. Liu, P. R. Reeves, Escherichia coli K12 regains its O    antigen. Microbiology 140 (Pt 1), 49-57 (1994); published online    Epub January (-   16. X. Zogaj, M. Nimtz, M. Rohde, W. Bokranz, U. Romling, The    multicellular morphotypes of Salmonella typhimurium and Escherichia    coli produce cellulose as the second component of the extracellular    matrix. Mol Microbiol 39, 1452-1463 (2001); published online Epub    March (-   17. E. Hochuli, W. Bannwarth, H. Dobeli, R. Gentz, D. Stuber,    Genetic Approach to Facilitate Purification of Recombinant Proteins    with a Novel Metal Chelate Adsorbent. 6, 1321-1325 (1988).-   18. S. N. Kim, Z. Kuang, J. M. Slocik, S. E. Jones, Y. Cui, B. L.    Farmer, M. C. McAlpine, R. R. Naik, Preferential binding of peptides    to graphene edges and planes. J Am Chem Soc 133, 14480-14483 (2011);    published online Epub September (10.1021/ja2042832).-   19. T. P. Hopp, K. S. Prickett, V. L. Price, R. T. Libby, C. J.    March, D. Pat Cerretti, D. L. Urdal, P. J. Conlon, A Short    Polypeptide Marker Sequence Useful for Recombinant Protein    Identification and Purification. Nat Biotech 6, 1204-1210 (1988).-   20. M. J. Pender, L. A. Sowards, J. D. Hartgerink, M. O.    Stone, R. R. Naik, Peptide-mediated formation of single-wall carbon    nanotube composites. Nano Lett 6, 40-44 (2006); published online    Epub January (10.1021/n1051899r).-   21. J. M. Slocik, M. O. Stone, R. R. Naik, Synthesis of gold    nanoparticles using multifunctional peptides. Small 1, 1048-1052    (2005); published online Epub November (10.1002/sm11.200500172).-   22. W. J. Chung, K. Y. Kwon, J. Song, S. W. Lee, Evolutionary    screening of collagen-like peptides that nucleate hydroxyapatite    crystals. Langmuir 27, 7620-7628 (2011); published online Epub June    (10.1021/1a104757 g).-   23. E. E. Oren, C. Tamerler, D. Sahin, M. Hnilova, U. O. Seker, M.    Sarikaya, R. Samudrala, A novel knowledge-based approach to design    inorganic-binding peptides. Bioinformatic 23, 2816-2822 (2007);    published online Epub November (10.1093/bioinformatics/btm436).-   24. B. Zakeri, J. O. Fierer, E. Celik, E. C. Chittock, U.    Schwarz-Linek, V. T. Moy, M. Howarth, Peptide tag forming a rapid    covalent bond to a protein, through engineering abacterial adhesin.    Proc Natl Acad Sci USA 109, E690-697 (2012); published online Epub    March (1115485109 [pii]10.1073/pnas.1115485109).-   25. C. L. Giltner, E. J. van Schaik, G. F. Audette, D. Kao, R. S.    Hodges, D. J. Hassett, R. T. Irvin, The Pseudomonas aeruginosa type    IV pilin receptor binding domain functions as an adhesin for both    biotic and abiotic surfaces. Mol Microbiol 59, 1083-1096 (2006);    published online Epub February (MMI5002    [pii]10.1111/j.1365-2958.2005.05002.x).-   26. W. Zhou, D. T. Schwartz, F. Baneyx, Single-pot biofabrication of    zinc sulfide immunoquantum dots. J Am Chem Soc 132, 4731-4738    (2010); published online Epub April (10.1021/ja909406n).-   27. M. E. Houston, H. Chao, R. S. Hodges, B. D. Sykes, C. M.    Kay, F. D. Sönnichsen, M. C. Loewen, P. L. Davies, Binding of an    oligopeptide to a specific plane of ice. J Biol Chem 273,    11714-11718 (1998); published online Epub May (-   28. A. Arakaki, J. Webb, T. Matsunaga, A novel protein tightly bound    to bacterial magnetic particles in Magnetospirillum magneticum    strain AMB-1. J Biol Chem 278, 8745-8750 (2003); published online    Epub March (10.1074/jbc.M211729200).-   29. J. D. Taylor, Y. Zhou, P. S. Salgado, A. Patwardhan, M.    Mcguffie, T. Pape, G. Grabe, E. Ashman, S. C. Constable, P. J.    Simpson, W.-C. Lee, E. Cota, M. R. Chapman, S. J. Matthews, Atomic    Resolution Insights into Curli Fiber Biogenesis. Structure 19,    1307-1316 (2011); published online Epub September 01    (10.1016/j.str.2011.05.015).-   30. O. Vidal, R. Longin, C. Prigent-Combaret, C. Dorel, M.    Hooreman, P. Lejeune, Isolation of an Escherichia coli K-12 mutant    strain able to form biofilms on inert surfaces: involvement of a new    ompR allele that increases curli expression. J Bacteriol 180,    2442-2449 (1998); published online Epub May (-   31. B. Zakeri, J. O. Fierer, E. Celik, E. C. Chittock, U.    Schwarz-Linek, V. T. Moy, M. Howarth, Peptide tag forming a rapid    covalent bond to a protein, through engineering a bacterial adhesin.    Proceedings of the National Academy of Sciences 109, E690-697    (2012); published online Epub April 20 (10.1073/pnas.1115485109).

CsgA polypeptide NCBI Ref Seq: NP_415560 SEQ ID NO: 1   1mkllkvaaia aivfsgsala gvvpqygggg nhggggnnsg pnselniyqy gggnsalalq  61tdarnsdlti tqhgggngad vgqgsddssi dltqrgfgns atldqwngkn semtvkqfgg 121gngaavdqta snssvnvtqv gfgnnatahq y nucleic acid sequence of CsgA-SpyTagSEQ ID NO: 2ATGAAACTTTTAAAAGTAGCAGCAATTGCAGCAATCGTATTCTCCGGTAGCGCTCTGGCAGGTGTTGTTCCTCAGTACGGCGGCGGCGGTAACCACGGTGGTGGCGGTAATAATAGCGGCCCAAATTCTGAGCTGAACATTTACCAGTACGGTGGCGGTAACTCTGCACTTGCTCTGCAAACTGATGCCCGTAACTCTGACTTGACTATTACCCAGCATGGCGGCGGTAATGGTGCAGATGTTGGTCAGGGCTCAGATGACAGCTCAATCGATCTGACCCAACGTGGCTTCGGTAACAGCGCTACTCTTGATCAGTGGAACGGCAAAAATTCTGAAATGACGGTTAAACAGTTCGGTGGTGGCAACGGTGCTGCAGTTGACCAGACTGCATCTAACTCCTCCGTCAACGTGACTCAGGTTGGCTTTGGTAACAACGCGACCGCTCATCAGTACGGCAGCGGTGGTTCTGGCGCGCACATCGTTATGGTTGACGCGTACAAACCGACCAAATGA amino acid sequence of CsgA-SpyTagSEQ ID NO: 3MKLLKVAAIAAIVFSGSALAGVVPQYGGGGNHGGGGNNSGPNSELNIYQYGGGNSALALQTDARNSDLTITQHGGGNGADVGQGSDDSSIDLTQRGFGNSATLDQWNGKNSEMTVKQFGGGNGAAVDQTASNSSVNVTQVGFGNNATAHQYGSGGSGAHIVMVDAYKPTKnucleic acid sequence of Amylase-SpyCatcher SEQ ID NO: 4ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCAAATCTTAATGGGACGCTGATGCAGTATTTTGAATGGTACATGCCCAATGACGGCCAACATTGGAAGCGCTTGCAAAACGACTCGGCATATTTGGCTGAACACGGTATTACTGCCGTCTGGATTCCCCCGGCATATAAGGGAACGAGCCAAGCGGATGTGGGCTACGGTGCTTACGACCTTTATGATTTAGGGGAGTTTCATCAAAAAGGGACGGTTCGGACAAAGTACGGCACAAAAGGAGAGCTGCAATCTGCGATCAAAAGTCTTCATTCCCGCGACATTAACGTTTACGGGGATGTGGTCATCAACCACAAAGGCGGCGCTGATGCGACCGAAGATGTAACCGCGGTTGAAGTCGATCCCGCTGACCGCAACCGCGTAATTTCAGGAGAACACCCAATTAAAGCCTGGACACATTTTCATTTTCCGGGGCGCGGCAGCACATACAGCGATTTTAAATGGCATTGGTACCATTTTGACGGAACCGATTGGGACGAGTCCCGAAAGCTGAACCGCATCTATAAGTTTCAAGGAAAGGCTTGGGATTGGGAAGTTTCCAATGAAAACGGCAACTATGATTATTTGATGTATGCCGACATCGATTATGACCATCCTGATGTCGCAGCAGAAATTAAGAGATGGGGCACTTGGTATGCCAATGAACTGCAATTGGACGGTTTCCGTCTTGATGCTGTCAAACACATTAAATTTTCTTTTTTGCGGGATTGGGTTAATCATGTCAGGGAAAAAACGGGGAAGGAAATGTTTACGGTAGCTGAATATTGGCAGAATGACTTGGGCGCGCTGGAAAACTATTTGAACAAAACAAATTTTAATCATTCAGTGTTTGACGTGCCGCTTCATTATCAGTTCCATGCTGCATCGACACAGGGAGGCGGCTATGATATGAGGAAATTGCTGAACGGTACGGTCGTTTCCAAGCATCCGTTGAAATCGGTTACATTTGTCGATAACCATGATACACAGCCGGGGCAATCGCTTGAGTCGACTGTCCAAACATGGTTTAAGCCGCTTGCTTACGCTTTTATTCTCACAAGGGAATCTGGATACCCTCAGGTTTTCTACGGGGATATGTACGGGACGAAAGGAGACTCCCAGCGCGAAATTCCTGCCTTGAAACACAAAATTGAACCGATCTTAAAAGCGAGAAAACAGTATGCGTACGGAGCACAGCATGATTATTTCGACCACCATGACATTGTCGGCTGGACAAGGGAAGGCGACAGCTCGGTTGCAAATTCAGGTTTGGCGGCATTAATAACAGACGGACCCGGTGGGGCAAAGCGAATGTATGTCGGCCGGCAAAACGCCGGTGAGACATGGCATGACATTACCGGAAACCGTTCGGAGCCGGTTGTCATCAATTCGGAAGGCTGGGGAGAGTTTCACGTAAACGGCGGGTCGGTTTCAATTTATGTTCAAAGAGGCGGCGGTTCTGATTACGACATCCCAACGACCGAAAACCTGTATTTTCAGGGCGCCATGGTTGATACCTTATCAGGTTTATCAAGTGAGCAAGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAGGACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGATTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTC G AAACCGCAGCACCAGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGCAAAGCAACTAAAGGTGACG CTCATATTTAAamino acid sequence of Amylase-SpyCatcher SEQ ID NO: 5MGSSHHHHHHSSGLVPRGSHMANLNGTLMQYFEWYMPNDGQHWKRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVISGEHPIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNENGNYDYLMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNGTVVSKHPLKSVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQRGGGSDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI

1. An engineered CsgA polypeptide, comprising a CsgA polypeptide with aC-terminal display tag flanking the CsgA polypeptide; wherein thedisplay tag comprises an activity polypeptide and a linker sequence;wherein the linker sequence is located N-terminal to the displaypolypeptide; and wherein the linker sequence comprises at least 6 aminoacids.
 2. The polypeptide of claim 1, wherein the linker sequenceconsists of glycine and serine residues.
 3. The polypeptide of claim 1,wherein the display tag and/or the activity polypeptide comprises apolypeptide selected from the group consisting of: Metal binding domain(MBD); SpyTag; graphene binding (GBP); carbon nanotube binding (CBP);gold binding (A3); CT43; FLAG; Z8; E14; QBP1; CLP12; and AFP8.
 4. Thepolypeptide of claim 1, wherein the activity polypeptide comprises aconjugation domain.
 5. The polypeptide of claim 4, wherein theconjugation domain is selected from the group consisting of: SpyTag;biotin acceptor peptide (BAP); biotin carboxyl carrier protein (BCCP);and a peptide comprising a LPXTG motif.
 6. A nucleic acid sequenceencoding the polypeptide of claim
 1. 7. A vector comprising the nucleicacid sequence of claim
 6. 8. An engineered microbial cell comprising thepolypeptide of claim
 1. 9. The cell of claim 8, wherein the cellexpresses an engineered CsgA polypeptide comprising an activitypolypeptide comprising a conjugation domain.
 10. The cell of claim 9,wherein the cell further comprises a nucleic acid sequence encoding afunctionalizing polypeptide comprising a partner conjugation domain. 11.A population of cells comprising a first cell type and a second celltype, wherein the first cell type is a cell of claim 9 and the secondcell type comprises a nucleic acid sequence encoding a functionalizingpolypeptide comprising a partner conjugation domain.
 12. A biofilmcomprising the cell of claim
 8. 13. A biofilm produced by culturing thecells of claim 8 under conditions suitable for the production of abiofilm.
 14. The biofilm of claim 13, comprising the cell of claim 8.15. A composition comprising the polypeptide of claim
 1. 16. Thecomposition of claim 15, wherein the composition comprises filamentscomprising the polypeptide of claim
 1. 17. The composition of claim 15,comprising a proteinaceous network.
 18. The composition of claim 15,wherein the composition further comprises additional proteinaceousbiofilm components
 19. The composition of claim 15, further comprisingthe cell of claim
 8. 20. The use of the cell of claim 8 to display apolypeptide within the biofilm, with the composition, or on the cellsurface.
 21. The use of the cell of claim 8 in an application selectedfrom the group consisting of: biocatalysis; industrial biocatalysis;immobilized biocatalysis; chemical production; filtration; isolation ofmolecules from an aqueous solution; water filtration; bioremediation;nanoparticle synthesis; nanowire synthesis; display of optically activematerials; biosensors; surface coating; therapeutic biomaterial;biological scaffold; structural reinforcement of an object; and as adelivery system for therapeutic agents.